Compositions and methods to modulate cell activity

ABSTRACT

The present invention provides methods and compositions for the remote control of cell function based on the use of radiofrequency waves to excite nanoparticles targeted to specific cell types. The nanoparticles may be applied to the target cell extracellularly and/or expressed intracellularly. The cell type of interest expresses a temperature sensitive channel wherein excitation of the nanoparticles results in a localized temperature increase that is transduced into a cellular response. Such cellular responses may include, for example, increases in gene expression resulting in production of one or more physiologically active proteins. The expression of such proteins can be used to treat a variety of different inherited or acquired diseases or disorders in a subject. Accordingly, the invention provides a generic approach for treatment of any disease associated with a protein deficiency.

The invention disclosed herein was made with United States Governmentsupport under NIH Grant No. R01 GM095654 from the National Institutes ofHealth. Accordingly, the United States Government has certain rights inthis invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a national phase application based on the PCT InternationalPatent Application No. PCT/US2012/052391, filed on Aug. 24, 2012, theentire contents of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides methods and compositions for the remotecontrol of cell function based on the use of radiofrequency waves toexcite nanoparticles targeted to specific cell types. The cell type ofinterest expresses a temperature sensitive channel wherein excitation ofthe nanoparticles results in a localized temperature increase that istransduced into a cellular response. Such cellular responses mayinclude, for example, increases in gene expression resulting inproduction of one or more physiologically active proteins. Theexpression of such proteins can be used to treat a variety of differentinherited or acquired diseases or disorders in a subject. Accordingly,the invention provides a generic approach for treatment of any diseaseassociated with a protein deficiency.

BACKGROUND OF THE INVENTION

The tools for dissecting the contribution of specific cells tophysiological functions and particular behavior have evolved over recentyears. Initial studies used electrical and chemical lesions to ablateboth neurons and fibers in defined regions. Later investigations madeuse of direct stimulation through implanted electrodes, however, thesestudies were hampered by variable activation, the need for permanentimplants, and tissue damage. As an alternative to these approaches,recent techniques make use of drug inducible systems to alter geneexpression or ion channels to modulate cell activity (Lerchner et al.,Neuron 54:35-49). By allowing the selective passage of cations oranions, families of ion channels regulate intracellular ionconcentrations, which in turn modulate intracellular functions accordingto the cell type. The use of ion channels has many advantages; theirstructure and function are relatively well described; they have a rapidtime course of activation, and a broad range of channels exist inmammalian and non-mammalian cells, which may be exploited in the searchfor the optimum means of modifying cellular activity. This approach wasfirst validated by transgenic expression of a drug-gated channel tomodify behavior, however, the time course of effects was relatively slow(hours to days) due to irreversible effects of the ligand. Recently, thenon-mammalian channelrhodopsin (ChR2) gene, a light activated cation,has been employed to rapidly activate molecularly defined neurons whenexposed to blue light (Boyden E S et al. 2005 Nat Neurosci 8:1263-1268).This system gives anatomical specificity and temporal control but alsohas limitations. For example, there are only two variants foractivation, thereby limiting the potential for combinatorial activation,and more importantly, activation in vivo requires fiber optic lightdelivery via implanted devices that are invasive and can interfere withbehavior.

The present invention provides methods and compositions for the remotecontrol of cell function based on the use of radiofrequency waves toexcite nanoparticles targeted to specific cell types. The invention,uses Nanoparticle Induced Circuit Excitation (NICE) to, for example,regulate ion channels as a means for stimulating the activity ofspecific cells remotely and non-invasively.

SUMMARY OF THE INVENTION

The invention described herein utilizes Nanoparticle Induced CircuitExcitation (NICE), which encompasses compositions and methods that havebeen developed for stimulating the activity of specific cells remotelyand non-invasively. The present invention provides methods andcompositions based on the use of radiofrequency waves to excitenanoparticles targeted to specific cell types. The cell type of interestexpresses a temperature sensitive channel wherein excitation of thenanoparticles results in a localized temperature increase that istransduced into a cellular response. The excitation of the nanoparticlesresults in a localized temperature increase that is transduced into acellular response such as, for example, an increase in gene expression.Such increases in gene expression may result in production of one ormore physiologically active proteins. The expression of such proteinscan be used to treat a variety of different inherited or acquireddiseases or disorders in a subject.

According to one aspect, the described invention provides a method toremotely stimulate the activity of a cell type of interest wherein thenanoparticles are externally applied. Such a method comprises: (i)administering to a cell population nanoparticles selective for the celltype of interest; and (ii) applying a radiofrequency field to remotelyactivate the nanoparticles. Said activation of the nanoparticles resultsin stimulation of the activity of the cell type of interest.

Alternatively, cells may be engineered to synthesize nanoparticlesintracellularly. For example, as described herein, the iron storageprotein ferritin, which forms a naturally occurring iron nanoparticle,was modified to form a ferritin fusion protein composed of a ferritinlight chain fused to ferritin heavy chain with a flexible linker region.Heating of the iron core by a RF magnetic field opens the TRPV1 channelto trigger calcium entry, increasing proinsulin gene expression andtriggering insulin release in vitro. This results in decrease bloodglucose in vivo.

In another embodiment of the invention as described herein, modificationusing intracellular nanoparticles uses a modified TRPV1 with a camelidantibody to GFP fused to the N-terminal of TRPV1 and a modified ferritinfusion protein with EGFP fused to the N-terminal of ferritin lightchain-linker-ferritin heavy chain. Heating of the iron core of theferritin attached to the TRPV1 triggers calcium entry and increasesproinsulin gene expression and proinsulin release in vitro.

In a non limiting embodiment of the invention, said nanoparticles may beparamagnetic nanoparticles.

According to another aspect of the invention, a method is provided toremotely stimulate the activity of a cell type of interest in a subject,the method comprising: (i) administering to the subject nanoparticlesselective for the cell type of interest; and (ii) applying aradiofrequency field to remotely activate the nanoparticles. Saidactivation of the nanoparticles results in stimulation of the activityof the cell type of interest in a subject.

Activities of the cell that may be stimulated include, for example,cellular responses such as cell proliferation and/or differentiation,apoptosis, activation of signal transduction pathways, neuronalactivation, development of long term potentiation and/or regulation ofgene expression.

Further, the invention provides a method to stimulate the activity of acell type of interest in a subject, the method comprising steps: (a)administering to the subject modified cells of interest that comprisenanoparticles that are selective for the cell type of interest; and (b)applying a radiofrequency field to remotely activate the nanoparticles.Said activation of the nanoparticles results in stimulation of theactivity of the cell type of interest in a subject.

The present invention can be used in a variety of different clinicalsettings. For example, the technology can be used to control theexpression of physiologically active proteins for used in treatment ofvarious inherited or acquired disorders or diseases. For example, stemcells, such as induced pluripotent stem cells (iPSC) or autologousmesenchymal stem cells engineered to express NICE constructs could actas autografts enabling external control of cell function. NICE dependentcalcium entry can then be used to regulate functions including hormonerelease, muscle contraction, or neural activity. Regulated hormoneexpression and release can facilitate the treatment of several endocrineconditions such as diabetes. Neuronal stimulation can be usedtherapeutically in several debilitating conditions such as Parkinson'sdisease (subthalamic stimulation) and stroke (transcranial directcurrent stimulation), as well as for pain relief and gastroparesis(Benabid A L. et al, 2009 Lancet Neurol 8:67-81; Schlaug G. et al. 2008Arch Neurol 65:1571-1576; Nnoaham K E, Kumbang J 2008 Cochrane DatabaseSyst RevCD003222; Maranki J, Parkman H P 2007 Curr Gastroenterol Rep9:286-294).

Functional nanoparticles, prepared using methods known to those skilledin the art, can be targeted by coating with recombinant antibodiesdirected to endogenous cell specific surface proteins. Theseapplications and the approaches can be applied in animals using the NICEtechniques.

Further, the methods and compositions of the invention provide a meansfor dissecting the contributions of defined cell populations tophysiology. The present invention makes it possible to decoratedifferent cell types with nanoparticles tuned to different frequencies,thus allowing one to simultaneously activate ensembles of defined cellseven if they are in proximity. The described invention provides forselective modification of cellular function non-invasively both in vitroand in vivo. At present, there are no methods for anatomically discrete,temporally controlled, non-invasive cell activation. Such a techniqueallows one to study the roles of cell populations in physiologicalprocesses, in particular those functions that are, or would be,perturbed by invasive methods.

Further, the invention proves non-human transgenic animals containingdifferent cell types that can be activated remotely through thetargeting of nanoparticles to the surface of said cells. The transgenicanimals provide an in vivo means for studying the contributions ofdefined populations of cells to physiology. Further, the transgenicanimals of the invention may be used as animal model systems for thescreening, identification and testing of useful therapeutic compounds.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals.

The described invention provides, for example, methods to remotelymodulate cell function in vertebrates and apply NICE to (i) modifyglucose metabolism (ii) activate dopaminergic neurons in the midbrainthat control reward and (iii) use a combinatorial activation scheme toregulate feeding behavior.

As described in detail below, a specific embodiment of the inventionmakes use of a unique combination of four components: (i) aradiofrequency electromagnetic field; (ii) cell-specific expression of ananoparticle tether; (iii) metallic/metal oxide nanoparticles; and (iv)a temperature sensitive TRPV cation channel to induce a tunable increasein intracellular calcium.

The present invention also provides pharmaceutical compositionscomprising nanoparticles that are selective for a cell type expressing atemperature sensitive channel. Alternatively, pharmaceuticalcompositions of the invention may comprise modified cells expressing atemperature sensitive channel of interest and decorated withnanoparticles selective for said cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Nanoparticles induced cell excitation to increase insulinexpression and release in vitro. Schema of nanoparticle-induced cellactivation and gene expression. Antibody-coated ferrous oxidenanoparticles bind to a unique epitope, His×6, in the firstextracellular loop of the temperature-sensitive TRPV1 channel. Exposureto a RF field induces local nanoparticle heating, which openstemperature-sensitive TRPV1 channels. Calcium entry triggers downstreampathways, such as activation of calcineurin, leading todephosphorylation of NFAT and translocation to the nucleus. Here, NFATbinds to upstream response elements to initiate gene expression of abioengineered human insulin gene. Additional calcium-dependent signaltransduction pathways also stimulate gene expression via binding to SREand CRE. P indicates a phosphate group.

FIG. 2. Heating of iron oxide nanoparticles in RF magnetic field. (A)Bulk heating effects of treating iron oxide nanoparticle suspensions (1mg/ml, 10-50 nm) in water with 465 kHz RF magnetic field. (B) TEM ofOcean Nanotech (SHP-20-50) iron oxide nanoparticles and their sizedistribution, calculated to be 19.83±2.7 from 450 particles. (C) X-rayphotoelectron spectroscopy of iron oxide nanoparticle samples. Surveyindicated the presence of iron, carbon, and oxygen with iron contentinvestigated in upper inset and the presence of carboxyl groupsconfirmed in the lower inset. (D) XRD pattern of iron oxidenanoparticles compared with JCPDS patterns #39-1346 (γ-Fe₂O₃) and#75-0033 (Fe₃O₄). (E) Bulk heating effects of treating iron oxidenanoparticle suspensions (1 mg/ml, 10-30 nm) in water with 13.56 Mhz,200 W, RF magnetic field.

FIG. 3. Nanoparticle decoration of cells in vitro. (A) Nanoparticledecoration of cells in vitro. Significant nanoparticle binding to thesurface of HEK293T cells expressing His tagged TRPV1 compared tountransfected cells. (B) Electron micrograph of anti-His antibody coatediron oxide nanoparticles (20 nm) binding to untransfected HEK293T cells(left panel) and HEK293 cells transfected with TRPV1^(His) (rightpanel). Scale bar 200 nm. (C) Immunoelectron micrography of anti-Hisantibody coated iron oxided nanoparticles (20 nm) co-localized withsilver enhanced gold immunostaining for TRPV1 (10 nm particles) intransfected HEK 293T cells (left panel). There is no TRPV1immunostaining in the absence of the primary antibody (right panel).Scale bar as indicated. (D) Representative changes in Fluo-4fluorescence after application of TRP agonist 2APB or RF treatment inHEK 293T cells transfected with TRPV1^(His) and decorated withnanoparticles.

FIG. 4. Temperature dependent release of proinsulin. Proinsulin releasefrom HEK 293T cells transfected with calcium dependent insulin alone,TRPV1^(His) and calcium dependent insulin or TRPV1 and calcium dependentinsulin was examined at 32° C., below the threshold for TRPV1 activationand at 44° C., above the threshold for TRPV1 activation. Expression ofTRPV1^(His) and TRPV1 significantly increased proinsulin release at 44°C. compared to that from cells without TRPV1. There is no significantdifference in the proinsulin release seen with TRPV1^(His) compared tounmodified TRPV1. (Same letter indicates p<0.05).

FIG. 5. (A) Bioengineered human insulin construct. Calcium dependentinsulin release is via three calcium response elements: serum responseelement (SRE), cyclic AMP response element (CRE) and nuclear factor ofactivated T-cell response element (NFAT RE) and a minimal promoterupstream of a furin sensitive human insulin cDNA. (B) RF treatment doesnot change proinsulin release from cells expressing the calciumdependent insulin gene from cells with TRPV1^(His), calcium dependenthuman insulin and nanoparticles in the absence of RF treatment, fromcells expressing TRPV1 and calcium dependent human insulin treated withRF but in the absence of nanoparticles, or from cells treated with RFexpressing calcium dependent human insulin and binding nanoparticles viaa nanoparticle tether comprised of a platelet derived growth factorreceptor transmembrane domain with fused extracellular biotin acceptor[protein but in the absence of TRPV1. (C) The effects of nanoparticleheating are cell specific. Cells transfected with a nanoparticle tether,BAPTM, and mixed with cells transfected with TRPV1 and calcium sependenthuman insulin show no increase in proinsulin release with RF treatment.(D) Translocation of NFAT1 with RF treatment. HEK cells transfected withTRPV1^(His)/calcium dependent human insulin incubated with anti-His ironoxide nanoparticles show almost exclusively cytoplasmic NFATimmunostaining without RF treatment (control, upper panels). RFtreatment results in NFAT staining in both the cytoplasm and nucleus ofthe cells (RF, lower panels). (E) Effect of calcineurin inhibitor on RFdependent proinsulin release. Proinsulin release from RF treated HEKcells transfected with TRPV1^(His)/calcium dependent human insulinincubated with anti-His oxide nanoparticles was blocked by preincubationwith Tacrolimus (100 nM). There is no difference apoptotic cellsincubated with increasing concentrations of nanoparticles as assessed byTUNEL count (F) are activated Caspase-3 count (G) between untreated andRF treated cells is transfected with TRPV1^(His).

FIG. 6. (A) RF treatment increases proinsulin release and insulin geneexpression in vitro. Nanoparticle-decorated HEK293T cells transfectedwith TRPV1^(His) and calcium-dependent insulin show a significantincrease in proinsulin release and insulin gene expression with RFtreatment that is blocked by the TRP antagonist ruthenium red. (Columnsmarked with the same letter indicate significance, P<0.05. Error barsindicate SEM) (B) Time sources of proinsulin release and insulin geneexpression from nanoparticle-decorated HEK293T cells transfected withTRPV1^(His) and calcium-dependent insulin with RF treatment.

FIG. 7 Expression of constructs and RF dependent proinsulin release fromES cells. (A) Expression of insulin in ES cell clones: Quantitative PCRmeasured expression of human insulin in 3 ES cell clones (4, 6 and 7)electroporated with TRPV1^(His) and Ca²⁺-dependent human insulinconstruct along with cells stably expressing TRPV1 alone, Ca²⁺-dependenthuman insulin construct alone or wild-type ES cells. (B) Expression ofTRPV1 in ES cell clones: Quantitative PCR measured expression of TRPV1in 3 ES cell clones (4, 6 and 7) electroporated with TRPV1^(His) andCa²⁺-dependent human insulin construct along with cells stablyexpressing TRPV1 alone, Ca²⁺-dependent human insulin construct alone orwild-type ES cells. (C) Immunohistochemistry for TRPV1 (upper panels) orHis (lower panels) in wild-type cells (left panels), cells stablyexpressing TRPV1^(His) (middle panels) or ES clone 7 (right panels). (D)RF dependent pro insulin release from ES cells. ES clone 7 expressingTRPV1^(His) and Ca²⁺-dependent human insulin incubated with iron oxidenanoparticles show a significant increase in proinsulin release inresponse to RF treatment (same letter indicates p<0.05).

FIG. 8. depicts dual component system for cell activation. (A) Schema ofdual component system. Streptavidin coated iron oxide nanoparticles bindbiotin on a cell surface biotin acceptor protein fused to atransmembrane domain (BAPTM). Exposure to an RF field induces localheating, which opens TRPV1 channels. Calcium entry triggers downstreamprocesses as before. (B) Nanoparticle binding to the surface of HEK 293T cells expressing TRPV1 and BAPTM is increased compared tountransfected cells (p=0.09). (C) Representative changes in Fluo-4fluorescence after application of TRP agonist 2APB or RF treatment innanoparticle decorated HEK 293 T cells transfected with dual componentsystem. (D) RF treatment increases proinsulin release in vitro from HEK293T cells transfected with TRPV1, BAPTM and calcium dependent humaninsulin. This is blocked by Ruthenium red. (Same letter indicatesp<0.05). (E) RF treatment increases insulin gene expression in vitro incells with TRPV1, BAPTM and calcium dependent human insulin and isblocked by Ruthenium red. (F) Time course of proinsulin release with RFtreatment from HEK293T cells transfected with TRPV1, BAPTM and calciumdependent human insulin. (G) Time course of insulin gene expression withRF treatment in cells transfected with TRPV1, BAPTM and calciumdependent human insulin.

FIG. 9. In vitro and in vivo studies on PC12 TRPV1^(His)/insulin stablecell line. (A) Proinsulin release from PC 12 cells stably expressingTRPV1^(His) and calcium dependent human insulin was significantlyincreased by a temperature above the threshold for TRPV1 activation(Same letter indicates p<0.05). (B) RF treatment significantly increasedproinsulin release from PC12 cells stably expressing TRPV1^(His) andcalcium dependent human insulin. (Same letter indicates p<0.01). (C) RFtreatment significantly increases insulin gene expression in PC12 cellsstably expressing TRPV1^(His) and calcium dependent human insulin (Sameletter indicates p<0.05). (D) Time course of proinsulin release fromPC12 cells stably expressing TRPV1^(His) and calcium dependent humaninsulin. RF treatment for 15 minutes significantly increased proinsulinrelease. (E) Serial blood glucose measurement in nude mice injected withPC12 cells expressing TRPV1His and calcium dependent human insulin toform a subcutaneous tumor. (F) Immunohistochemistry for TRPV1 and Hisepitope tag in sections from tumors formed following subcutaneousinjection of PC12 cells stably expressing TRPV1^(His) and calciumdependent human insulin.

FIG. 10. Nanoparticle regulation of blood glucose in vivo. (A) Effectsof RF treatment on blood glucose in PBS and nanoparticle-treated micewith tumors expressing TRPV1^(His) and calcium-dependent human insulin.RF treatment significantly reduces blood glucose in nanoparticle-treatedmice compared with that of PBS-treated mice. (Asterisks indicate P<0.05.Error bars indicated SEM.) (B) RF treatment of mice with tumorsexpressing TRPV1^(His) and calcium-dependent human insulin injected withnanoparticles significantly reduces blood glucose over the course of thestudy as assessed by the area under the curve. There is no effect inmice with tumors expressing calcium-dependent insulin alone withoutTRPV1^(His) (same letter indicates P<0.05.) (C) Plasma insulin issignificantly increased by RF treatment in nanoparticle-treated but notPBS-treated mice with tumors expressing TRPV1^(His). (Same letterindicates P<0.05). (D) Insulin gene expression is significantlyincreased in the tumors expressing TRPV1^(His) and calcium-dependenthuman insulin treated with nanoparticles and RF magnetic field but notin tumors expressing calcium-dependent human insulin alone withoutTRPV1^(His).

FIG. 11. Nanoparticle regulation of blood glucose in vivo. (A) Protocolfor assessment of effects of RF treatment on blood glucose in micebearing tumors expressing TRPV1^(His) and calcium dependent humaninsulin. At time −30 min, mice are anesthetized and injected with PBS ornanoparticles. RF stimulation begins at time 0 and continues for 30mins. Mice remain anesthetized for a further 30 mins. Samples for plasmainsulin are taken at −30 and +30 mins and samples for blood glucose aretaken before, during and after RF stimulation. (B) Expression of c-fosgene in tumors showed no difference in levels between control(untreated) and RF treated tumors. (C) No increase in apoptotic cellsfrom nanoparticle and RF treated tumors as assessed by TUNEL. (D) Noincrease in apoptotic cells from nanoparticle and RF treated tumors asassessed by immunohistochemistry (IHC) for activated Caspase-3. (E)Effects of RF treatment on blood glucose in PBS and nanoparticle treatedmice with tumors expressing TRPV1^(His) and calcium dependent humaninsulin. RF treatment significant reduces blood glucose in nanoparticletreated mice compared to PBS treated mice in both the first and secondstudy separated by a week. (Asterisk indicates p<0.05.) (F) Cumulativeblood glucose change, measured by area under the curve, shows asignificant decrease in nanoparticle treated mice compared to PBStreated mice in both the first and second study. There is no significantdifference between the AUC for the nanoparticle studies.

FIG. 12. Effect of NICE in the absence of TRPV1, absence of anti-Hisantibodies and temperature studies in vivo. (A) Effect of RF stimulationon blood glucose in PBS and nanoparticle treated mice bearing tumorsexpressing calcium dependent insulin gene without TRPV1. (B) Effect ofRF stimulation on blood glucose in mice treated with PBS ornanoparticles which have not been conjugated to anti-His antibody. (C)Thermal imaging using an infrared camera on mouse with tumor expressingTRPV1^(His) and calcium dependent human insulin injected with iron oxidenanoparticles before (left panel) and after (right panel) RF magneticfield treatment. (D) Core body temperature and intra-tumor temperaturerecordings from mice with TRPV1^(His) expressing tumors followingnanoparticle injection and RF treatment. There is no difference in theintratumoral temperature achieved with RF treatment in the tumors ofmice with TRPV1^(His) and insulin expression compared to the tumors ofmice with insulin expression but without TRPV1^(His) expression.

FIG. 13. Intracellular nanoparticle synthesis and cell activation. (A)Schema of intracellular nanoparticle synthesis and cell activation. Aferritin fusion protein is composed of a ferritin light chain fused toferritin heavy chain with a flexible linker region. Heating of the ironcore by a RF magnetic field opens the TRPV1 channel to trigger calciumentry, as previously described. (B) RF treatment increases proinsulinrelease in vitro. HEK293 T cells transiently transfected with TRPV1,ferritin fusion protein, and calcium-dependent human insulin show asignificant increase in proinsulin release in response to RF treatment.(Same letter indicates significance, P<0.05.) RF treatment does notincrease proinsulin release from cells expressing ferritin in theabsence of TRPV1. (C) RF treatment increases insulin gene expression invitro. Insulin gene expression is significantly increased by RFtreatment in cells transfected with TRPV1, ferritin fusion protein, andcalcium-dependent human insulin. (Same letter indicates significance,P<0.05). RF treatment does not increase insulin gene expression in cellsexpressing ferritin fusion protein in the absence of TRPV1.

FIG. 14. Expression of Ferritin fusion protein in vitro. (A) Ferritinexpression as shown by IHC for ferritin light chain. (B) Electronmicrograph of iron loaded ferritin in transfected cells. Scale bar 200nm.

FIG. 15. Release of proinsulin with RF (465 kHz) from 293 cellstransfected with TRPV1 and myristoylated ferritin fusion protein(mFerritin), transfected with TRPV1 with n-terminal fusion of camelidantibody to EGFP (vhh-TRPV1) and EGFP fused to ferritin fusion proteinand transfected with TRPV1 with n-terminal fusion of camelid antibody toEGFP (vhh-TRPV1), EGFP fused to ferritin fusion protein and camelidantibody fused to ferritin fusion protein along with calcium dependentinsulin gene.

FIG. 16. Release of proinsulin from (A) embryonic stem cells from C57BL6mice expressing TRPV1, myristoylated ferritin fusion protein and calciumdependent insulin, decorated with nanoparticles and treated with RF and(B) mesenchymal stem cells from C5BL6 mice expressing TRPV1,myristoylated ferritin fusion protein and calcium dependent insulin andtreated with RF.

FIG. 17. Regulation of blood glucose in wild-type mice. C57BL6 micereceived in injection of replication deficient adenovirus expressingTRPV1, myristoylated ferritin fusion protein (mferritin) and calciumdependent insulin or adenovirus expressing LacZ. Two weeks afterinjection, mice were fasted overnight and anesthetized then treated withRF for 1 hour and blood glucose monitored. RF treatment ofTRPV1/mferritin/calcium dependent insulin significantly reduced bloodglucose compared to baseline and compared to either RF treated LacZexpressing mice or mice expressing TRPV1/mferritin/calcium dependentinsulin without RF treatment.

FIG. 18 (A) Change in blood glucose and (B) change in blood glucoseexpressed as area under curve in nude mice injected with mesenchymalstem cells alone (control) and treated with RF or mesenchymal stem cellsexpressing TRPV1, mferritin and calcium dependent insulin and treatedwith RF.

FIG. 19. (A) A synthetic promoter comprised of three calcium responseelements: serum response element (SRE), cyclic AMP response element(CRE) and nuclear factor of activated T-cell response element (NFAT RE)and a minimal promoter were cloned upstream of a modified, furinsensitive insulin cDNA. HEK 293t cells expressing calcium dependenthuman insulin and either TRPV1His or TRPV1 BAP were decorated withfunctionalized IO nanoparticles. Applying a RF magnetic field tonanoparticle-decorated cells expressing TRPV1His or TRPV1 BAP andcalcium regulated furin sensitive insulin significantly increasedproinsulin release and insulin gene expression. The increases inproinsulin release are blocked by the non-specific TRP channelinhibitor, ruthenium red. There was a trend towards an increase inproinsulin release after 15 mins of RF treatment, presumably initiallythrough the release of preformed insulin containing vesicles and with asignificant increase in release at 1 hour (D), whilst insulin geneexpression begins to increase after 45 minutes and also becomessignificant at 1 hour (E). The effects of RF dependent heating of IOnanoparticles were confined to decorated cells since there was norelease of proinsulin when cells expressing BAPTM as a nanoparticletether are mixed with, and therefore adjacent to, cells expressing TRPV1and calcium regulated furin sensitive insulin. This time course issimilar to the expression of c-fos, a gene whose expression is alsocalcium dependent. To assess cell viability, immunohistochemistry wasused to quantify two markers of apoptosis—active caspase 3 and terminaldeoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Nodifferences in immunopositive cell counts were observed between TRPV1Histransfected 239t cells treated with nanoparticles alone and thosetreated with nanoparticles and RF magnetic field application (G and H).

FIG. 20 shows the effect of NICE in vivo. (A) Protocol to examine theeffect of RF on blood glucose and insulin in vehicle or nanoparticleinjected TRPV1/NFAT-insulin tumors in nude mice. (B) Effect of RF onblood glucose in vehicle (PBS) or nanoparticle injected mice. Asignificant difference in blood glucose is seen at 30, 45 and 60minutes. (C) Assessment of area under the curve for circulating bloodglucose shows a significant difference between PBS and nanoparticletreated groups between 0 and 120 minutes. (D) Circulating insulin levelsincrease significantly in nanoparticle treated mice. (E) Insulin geneexpression, as assessed by qPCR, is significantly increased in RFtreated tumors.

FIG. 21. shows a schema of intracellular nanoparticle synthesis usingferritin chimeras. (A) Iron binding chimeric ferritin peptides composedof ferritin light chain (FLC) and ferritin heavy chain (FHC) with aflexible linker sequence (pink box) are fused to either EGFP (green box)or the high affinity camelid anti-GFP antibody (yellow box). When theseare expressed they form ferritin complexes with either EGFP or nanobodyat the surface. The nanobody peptide is also fused to the intracellularC terminal of the temperature sensitive calcium channel, TRPV1.Expression of all three components in the cell results in an ironcontaining ferritin aggregate attached to the intracellular C terminalof TRPV1. (B) Electron microscopy image of chimeric ferritin complexes(arrows) in 293t cells transfected with egfp-chimeric ferritin andnanobody-chimeric ferritin. (C) Cell surface expression of HA-taggednanobody fused to TRPV1 in transfected 293t cells.

FIG. 22 shows heating of iron oxide nanoparticles in 465 kHzradiofrequency field. (A) Exposure of 20 nm ferrous oxide nanoparticlesuspension (1 mg/ml) and water to 465 kHz radiofrequency field, (B)Significant increase in temperature of nanoparticles (compared towater). Nanoparticle temperature increases by 5° C. in 30 s without anyincrease in water temperature.

FIG. 23 shows confirmation of co-expression of TRPV1 and nanoparticletether in vitro. (A) Dual staining for TRPV1 and HA (upper panels).TRPV1 and biotin (middle panels) and TRPV1 and streptavidin Alexa 594(lower panels) in transfected HEK 293t cells. (B) Streptavidin coatediron oxide nanoparticle binding (10 nm) to transfected HEK 293t cells(left) and quantification in non-transfected and transfected cells(right).

FIG. 24 shows opening of TRPV1 channels and calcium entry in response tonanoparticle heating by RF in vitro. (A) TRPV1 opening and rapid calciumentry in HEK293t cells transfected with TRPV1 and BAPTM and decoratedwith streptavidin coated nanoparticles in response to nanoparticleheating in RF field. Calcium entry was measured as a change influorescence intensity of the calcium indicator Fluo-4. (B)Pseudocolored images indicating change in fluorescence intensity inTRPV1 transfected cells with RF stimulation. (C) Indirect assessment ofintracellular calcium via expression of luciferase under the control ofa calcium dependent NFAT promoter. (D) Luciferase expression issignificantly increased in HEK293t cells only in the presence of allcomponents of the NICE system: TRPV1 and the biotin acceptor protein(BAP), the addition of 20 nm iron oxide nanoparticles (NP) and thepresence of 465 kHz, 110 kA/m electromagnetic field (RF).

FIG. 25 shows constructs for viral delivery of NICE components and RFdependent hormone release. (A) Construct for constitutive expression ofNICE components, BAPTM and TRPV1. (B) Construct for calcium dependentexpression of furin modified human insulin. (C) Construct inserted intoadenovirus for cre dependent expression of NICE components, BAPTM andTRPV1 using FLEX system. (D) 293t cells transiently transfected withTRPV1-Baptm and NFAT insulin show a significant increase in proinsulinrelease with nanoparticle binding and RF exposure. (E) Insulin geneexpression is also significantly increased in these cells with RFexposure.

FIG. 26 shows combinatorial activation of transfected cells. A mixtureof 2 cell populations will be studied each expressing a unique linearepitope in the first extracellular loop of TRPV1 to tether an antibodycoated nanoparticle tuned to a distinct wavelength. Subsequent calciumentry increase expression of a calcium dependent luciferase unique toeach cell population. (A) Iron oxide nanoparticles coated with anti-His6× antibody bind to His 6 epitope in TRPV1. Calcium entry triggers CBRluc expression. (B) Gold nanoparticles coated with anti-FLAG antibodybind to FLAG epitope in TRPV1. Calcium entry triggers CBG99 lucexpression.

FIG. 27 shows constructs for generation of transgenic mice forexpression of NICE components. Transgenic mice will be generated withthe insulin promoter driving expression of TRPV1 (A) and BAPTM (B).These mice are crossed to express both TRPV1 and BAPTM in beta cells(C). An additional transgenic mouse with luciferase downstream of NFATresponse elements will act as an in vivo reporter of intracellularcalcium (D). The resulting mice (E) will express TRPV1 and BAPTM in betacells and calcium dependent luciferase in all cells.

FIG. 28 shows an illustrative scheme for self-stimulation protocol withlickometer: (A) a fiber connector for implant of a biocompatible 200 μmfiber optic; (B) implanted fiber to deliver light to the ventraltegmental area; (C) Med associates photobeam lickometer; (D) theself-stimo-lick paradigm is a variation of the self-stimulation paradigmwhere the operant behavior is a lick. Light stimulation of ChR2 positiveneurons or RF stimulation of NICE positive neurons occurs only when themouse consumes water from the lickometer; (E) animals injected withAAV-Flex-hChR2-mCherry virus (top) consume more water than controls(bottom) form the connected port. (F) cumulative licks over a 2 hourtrial. Light sensitive (red) and control animals (blue) in a SSLparadigm.

FIG. 29 shows expression of NICE constructs in Agrp and POMC neurons.(A) generation of BAC transgenic mouse with His tagged TRPV1 under thecontrol of Agrp promoter; (B) transgenic mouse with Flag-tagged TRPV1under control of POMC promoter; (C) these transgenic lines are crossedto result in mice with His tagged TRPV1 expression in Agrp neurons andFlag tagged TRPV1 expression in POMC neurons of the arcuate nucleus; (D)without RF stimulation neither AGrp nor POMC neurons are activated; (E)in the presence of anti-His iron oxide beads and RF of 465 kHz,His-tagged Agrp neurons are activated; (F) in the presence of anti-FLAGgold nanorods and RF of 13.5 MHz, FLAG tagged POMC neurons areactivated; (G) with both anti-His iron oxide beads and anti-FLAG goldnanorods and RF of 13.5 MHz, both His tagged Agrp neurons and FLAGtagged POMC neurons are activated.

FIG. 30. Iron oxide (IO) nanoparticles functionalized with monoclonalantibodies against the His×6 epitope tag are targeted to cells. (A-B) Inthe presence of a RF field, local heating of IO nanoparticles above thethreshold for TRPV1 channel activation (42° C.) triggers calcium entryand cell activation.

FIG. 31. Depiction of a modified TRPV1 channel as both a nanoparticletether and effector. A short epitope tag, His×6, was introduced into thefirst extracellular loop of TRPV1. Modification did not significantlyalter the temperature sensing ability of TRPV1.

FIG. 32. No significant increase in proinsulin release was observed inHEK 293t cells decorated with IO nanoparticles in the presence of an RFmagnetic field without TRPV1 (transfected with BAP only), in cells withTRPV1 but without nanoparticle binding (transfected with TRPV1 only), orin cells with nanoparticles and TRPV1 (TRPV1^(His) or TRPV1 BAP) but inthe absence of the RF magnetic field.

FIG. 33. In vitro studies examining the effects of RF treatment onproinsulin release and insulin gene expression replicated the findingsin transfected HEK 239t cells (A-C). Stably transfectedPC12-TRPV1His-Ins cells were injected subcutaneously into the flank ofnude mice and formed tumors expressing TRPV1His (D) and furin sensitiveinsulin constructs. Following an overnight fast, PBS or IO nanoparticleswere injected into the tumors of anesthetized mice (50 ul total volume,nanoparticle concentration 8 mg/ml). Blood glucose and plasma insulinwere measured before, during and after the application of an RF field(E).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for the remotecontrol of cell function based on the use of radiofrequency waves toexcite nanoparticles targeted to specific cell types. The cell type ofinterest expresses a temperature sensitive channel wherein excitation ofthe nanoparticles results in a localized temperature increase that istransduced into a cellular response. Such cellular responses, includefor example, modulation of cell proliferation, cell differentiation,apoptosis, and/or activation of signal transduction pathways. In aspecific embodiment of the invention, the cellular response is anincrease in gene expression resulting in production of one or morephysiologically active proteins. The expression of such proteins may beused to treat various inherited or acquired disorders including forexample, cardiovascular disorders, central nervous system disorders,autoimmune diseases, oncological diseases, hormonal disorders, metabolicdiseases, blood disorders or immune disorders. Additionally, theproteins may be expressed to treat various infectious diseasesincluding, for example, viral, bacterial, parasitic, and fungalinfections. The cellular response resulting from nanoparticle excitationmay also be designed to result in an increase in gene expressionresulting in production of one or more nucleic acid molecules ofinterest. Such nucleic acid molecules include those molecules capable ofregulating protein expression, such as antisense and siRNA molecules.

The expression system of the present invention can be used withvirtually any type of biological cell population, including prokaryoticand eukaryotic cells. Such eukaryotic cells include, for example, plantand mammalian cells. The specific cell type used will typically varydepending upon the type of cellular response that is sought to beregulated. For example, mammalian cells and specifically, human cells oranimal cells are typically preferred for increased expression of aphysiological protein for use as a therapeutic.

In an embodiment of the invention the cell type of interest is a stemcell, preferably a mammalian stem cell. For example, stem cellsengineered to express NICE constructs can act as autografts to enableexternal control of cell function. As used herein, “stem cell” refers toany cell having the potential to differentiate into one or moredifferent cell types, including pluripotent stem cells. Such cellsinclude, but are not limited to, stem cells derived from a variety ofdifferent sources including, for example, bone marrow, embryonicblastocysts or yolk sac, spleen, blood, including peripheral blood andumbilical cord blood, adipose tissue and other tissues and organs. Suchstem cells include, but are not limited to, hematopoietic stem cells,mesenchymal stem cells, endothelial progenitor cells or embryonic stemcells.

In a specific embodiment of the invention, the cell type of interestexpresses a temperature sensitive channel wherein activation of thenanoparticles results in a localized temperature increase that istransduced into a cellular response via the temperature sensitivechannel. Such temperature sensitive channels include, but are notlimited to, TRPV1 TRPV2, TRPV3, TRPM8, chimeric TRP channels, and tandempore domain potassium channels, such as TREK1, TREK2, and TASK. Thelocalized temperature increase mediated by the excitation of thenanoparticles leads to an activation of the TRPV1 channel resulting ingating of Ca2+ entry.

In a specific embodiment of the invention, the cell type of interestexpresses a channel wherein activation of the nanoparticle results inmotion of the nanoparticle that is transduced into a cellular responsevia the mechanical motion of the particle. Such motion sensitivechannels include, but are not limited to, TREK-1, TRAAK, TRPV4, andTRPV1. The localized stimulation of nanoparticle motion leads to anactivation of the channel resulting in modulation of cell activity.

In an embodiment of the invention, the cells to be targeted may begenetically engineered to express one or more genes encodingphysiologically active proteins of interest, such as those proteinsproviding a therapeutic benefit. The cells are genetically engineered insuch a way that expression of the protein(s) of interest is induced inthe cell upon excitation of the nanoparticles which results in alocalized temperature increase or an increase in nanoparticle motion.Alternatively, the cells to be targeted may be engineered to express anon-encoding nucleic acid molecule of interest such as an antisense orsiRNA molecule. Additionally, the target cells maybe geneticallyengineered to express a temperature sensitive protein, such as atemperature sensitive ion channel, wherein an increase in temperaturemediated by the excited nanoparticles results in a cellular responsethrough activation of the ion channel.

In another embodiment of the invention, target cells may be engineeredto intracellularly express a protein that is capable of acting as anactivated nanoparticle upon exposure to a RF magnetic field. Suchproteins include for example, the iron storage protein ferritin. Suchproteins may be expressed in the cell as fusion proteins to target theirlocation to a specific site within the cell, for example, in closeproximity to a temperature sensitive channel.

In an embodiment of the invention, recombinant expression vectorsdesigned to express a physiologically active protein of interest, or anucleic acid molecule of interest, such as antisense or RNAi molecules,or a protein that may act as a natural nanoparticle can be introducedinto the target cells of choice.

The recombinant expression vectors, in addition to containing a nucleicacid encoding the protein or nucleic acid of interest, will containtranscriptional regulatory sequences that can be induced upon excitationof the particles resulting in expression of the protein, or nucleic acidmolecule of interest. Such transcriptional regulatory sequences,include, but are not limited to, promoter and/or enhancer sequences thatinduce gene expression in response to ion channel activation. Suchregulatory sequences include, but are not limited to the calciumresponse elements, referred to herein as SRE, CRE and NFAT RE.

The cells may be genetically engineered using techniques well known inthe art. Such techniques include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook J et al. 2000.Molecular Cloning: A Laboratory Manual (Third Edition), and Ausubel etal (1996) Current Protocols in Molecular Biology John Wiley and SonsInc., USA). Any of the methods available in the art for gene deliveryinto a host cell can be used according to the present invention todeliver genes into the target cell population. Such methods includeelectroporation, lipofection, calcium phosphate mediated transfection,or viral infection. In a specific embodiment, a viral vector thatcontains a nucleic acid encoding the protein or nucleic acid of interestand a transcriptional regulatory sequence that can be induced uponexcitation of the nanoparticles can be used. Such viral vectors includefor example, retroviral, adenoviral or adeno-associated viral vectors.(See, Kozarsky and Wilson, 1993, Current Opinion in Genetics andDevelopment 3:499-503 for a review of adenovirus-based gene delivery).

For general reviews of the methods of gene delivery see Strauss, M. andBarranger, J. A., 1997, Concepts in Gene Therapy, by Walter de Gruyter &Co., Berlin; Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 33:573-596; Mulligan, 1993, Science 260:926-932; and Morgan andAnderson, 1993, Ann. Rev. Biochem. 62:191-217; 1993, TIBTECH11(5):155-215.

In order to access different organs non-invasively, it is necessary tohave an electromagnetic field that is capable of passing through tissueas part of a system that allows some cells to be activated while themajority are not. Accordingly, radiofrequency (RF) electromagneticfields are used for this purpose. RF signals at low and mediumfrequencies penetrate tissues freely and without significant energyabsorption making it now possible to adapt this system for in vivo use(Jokela International Union of Radio Science 2008). In contrast totissues, metallic/metal oxide nanoparticles placed in an alternating RFfield absorb energy and heat in a controlled manner depending on thestrength of the field, a process known as induction heating (Fortin etal., J. Am, Chem. Soc. 129:2628-2635). The heating capacity depends onnanoparticle composition, size, perhaps shape and the frequency andpower of the RF field and, as such, it is possible to regulate the heatgenerated within the physiological temperature range.

In vitro, the temperature response achieved is fast and decays quickly(inverse of the square of the distance) thus providing a rapid,functional ‘on-off’ switch. The nanoparticles employed, for example,magnetic iron oxide and gold spheres, are easily prepared, have littleor no intrinsic cell toxicity and can readily be adapted to target cellsby incorporating streptavidin, antibodies, or pharmacological agents(Samanta B. et al. J Mater Chem 18:1204-1208; Wang A Z. et al. 2008Expert Opin Biol Ther 8:1063-1070). Therefore, they are well suited forinducing localized temperature changes that can be transduced intocellular responses in vitro and in vivo.

Nanoparticles of differing compositions and shapes are heated at definedrates by different electromagnetic field frequencies and strengths.Nanoparticles for use in the present invention include, but are notlimited to, metallic nanoparticles, and metal oxide nanoparticles. Suchnanoparticles include, but are not limited to, iron oxide nanoparticles,gold nanoparticles, and the like. For example, iron oxide nanoparticlesare maximally heated by an EM frequency of around 465 kHz while goldnanoparticles heat at an EM frequency of 13.5 MHz, with the fieldstrength determining the rate of heating. This property potentiallyallows distinct EM frequencies to differentially heat nanoparticles ofdifferent compositions and/or shapes. Nanoparticles consisting ofdifferent chemistries, such as, but not limited to, gold and iron oxide,and of different shapes, particularly nanoparticles of different aspectratios (e.g., spheres vs. rods), can be chosen based on their discreteheating frequencies and resistivities. The described invention providesnon-limiting, illustrative compositions and methods encompassingdifferent chemistries and spheres of specific sizes. The nanoparticlescan be directed to distinct cell populations via cell-specificexpression of unique tethers and then, using RF generators and tunableamplifiers with a range of excitation frequencies, excite different cellpopulations alone or in combination.

Further, the nanoparticles can be conjugated to various biological orchemical moieties that bind a specific receptor or target a specificcell type. In such instances, the nanoparticles may be externallyapplied to the cells. The ligand can comprise a small molecule, peptide,antibody, nucleic acid, protein, carbohydrate, lipid, polyethyleneglycol derivatives or any combination thereof. Metal nanoparticles canreadily be functionalized to target define cell populations by coatingwith specific antibodies that recognize proteins that are normallyexpressed on a cell or transfected into that cell (Samanta et al., J.Nat. Chem. 18:1204-1208; Wang et al., Expert. Opin. Biol. Ther.8:1063-1070). For example, streptavidin, which binds to biotin withextremely high affinity, may be conjugated to nanoparticles. Throughconjugation of strepavidin to nanoparticles, a system is providedwhereby streptavidin-conjugated nanoparticles can be coupled tobiotin-labeled cells through the strepavidin/biotin high-affinityreaction. Loading of nanoparticles onto the cells permits targeting ofheat to said cells.

In addition to external application of nanoparticles to the target cellof interest, cells may be genetically engineered to express proteinswhich can act as naturally occurring nanoparticles and which can beactivated by a RF magnetic field. Such proteins include, for example,the iron storage protein ferritin, the bacterial gene MagA,ceruloplasmin and transferrin.

The method of the present invention comprises contacting a target cellpopulation with nanoparticles for a time sufficient to permit binding ofthe nanoparticle to the surface of the target cell. In an embodiment ofthe invention, the nanoparticles are administered in vivo to a subjectresulting in contacting of a target cell population with nanoparticlesfor a time sufficient to permit binding of the nanoparticle to thesurface of the target cell.

In an embodiment of the invention, the target cells are cultured, usingroutine tissue culture methods well known to those of skill in the art.Cells are then washed with a buffer, such as a phosphate-buffered saline(PBS) and a solution of nanoparticles is added to the target cells. Inan embodiment of the invention, the nanoparticle solution comprises amixture of the tissue culture media in which the cells are cultured andnanoparticles. Cells are incubated with the nanoparticles for a timesufficient to permit efficient binding of the nanoparticles to thetarget cells. Transfer of nanoparticles to the target cells can bemonitored using, for example, flow cytometry.

In a preferred embodiment of the invention the target cell populationare stem cells. Also within the scope of the invention are nanoparticlelabeled cells that have been genetically engineered to express a desiredprotein, or nucleic acid of interest. For example, nanoparticle labeledcells may be engineered to express proteins capable of providing atherapeutic benefit.

The present invention further provides pharmaceutical compositionscomprising nanoparticles, nanoparticle-labeled cells and/or cells thatexpress nanoparticles intracellularly and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known to thoseskilled in the art and include, but are not limited to, 0.01-0.1M andpreferably 0.05M phosphate buffer, phosphate-buffered saline (PBS), or0.9% saline. Such carriers also include aqueous or non-aqueoussolutions, suspensions, and emulsions. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, saline andbuffered media. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate.

Nanoparticle-labeled cells and/or cells that express nanoparticlesintracellularly can also be incorporated or embedded within scaffoldswhich are recipient-compatible and which degrade into products which arenot harmful to the recipient. These scaffolds provide support andprotection for nanoparticle-labeled cells that are to be transplantedinto the recipient subjects.

The present invention provides methods and compositions which may beused to provide a therapeutic benefit for treatment of various diseases.Specifically, through the use of nanoparticle-labeled cells and/or cellsthat express nanoparticles intracellularly, a therapeutic protein, ornucleic acid molecule of interest, may be delivered to a subject in needof treatment through administration of nanoparticle labeled cells.Alternatively, nanoparticles themselves may be administered to a subjectin need of treatment, wherein said nanoparticles are targeted toendogenous cells of the subject wherein excitation of the nanoparticleresults in a localized temperature increase that is transduced into acellular response.

Various delivery systems are known and can be used to administer thenanoparticles, nanoparticle labeled cells and/or cells that expressnanoparticles intracellularly. Such compositions may be formulated inany conventional manner using one or more physiologically acceptablecarriers optionally comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

The methods of the invention, comprise administration of nanoparticlesand/or nanoparticle labeled cells and/or cells that intracellularlyexpress a nanoparticle, in a pharmaceutically acceptable carrier, fortreatment of various disorders or diseases. “Administering” shall meandelivering in a manner which is effected or performed using any of thevarious methods and delivery systems known to those skilled in the art.Administering can be performed, for example, pericardially,intracardially, subepicardially, transendocardially, via implant, viacatheter, intracoronarily, intravenously, intramuscularly,subcutaneously, parenterally, topically, orally, transmucosally,transdermally, intradermally, intraperitoneally, intrathecally,intralymphatically, intralesionally, epidurally, or by in vivoelectroporation. Administering can also be performed, for example, once,a plurality of times, and/or over one or more extended periods.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. The composition can be formulatedas a suppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carvers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the therapeutic compound,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The appropriate concentration of the compositions of the invention whichwill be effective in the treatment of a particular disorder or diseasewill depend on the nature of the disorder or disease, and can bedetermined by one of skill in the art using standard clinicaltechniques. In addition, in vitro assays may optionally be employed tohelp identify optimal dosage ranges. The precise dose to be employed inthe formulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses maybe extrapolated from dose response curves derivedfrom in vitro or animal model test systems. Additionally, theadministration of the compound could be combined with other knownefficacious drugs if the in vitro and in vivo studies indicate asynergistic or additive therapeutic effect when administered incombination.

Additionally, the progress of the recipient receiving the treatment maybe determined using assays that are designed to detect thephysiologically active protein expressed by the nanoparticle targetedcells.

The present invention further relates to transgenic non-human animalsthat may be engineered to contain cells that respond to nanoparticleexcitation in a desired fashion. For example the transgenic animals maybe engineered to express cell surface receptors that act as bindingpartners for the nanoparticles. Said target cells may either naturally,or through genetic engineering, express a protein, or nucleic acidmolecule of interest upon nanoparticle excitation. Alternatively, thetransgenic animals may be engineered to intracellularly express ananoparticle, such as for example a naturally occurring ironnanoparticle. Such transgenic animals provide in vivo model systems forstudying normal physiological processes as well as disease processes.The transgenic animals of the invention may further be useful as in vivomodel systems for use in identification and testing of novel therapeuticcompounds of interest.

According to one aspect of the described invention, the method of theinvention can be implemented as follows. First, expression of both abiotin acceptor protein (BAP) fused to the transmembrane domain ofplatelet derived growth factor receptor (PDGFR) as a tether forstreptavidin coated nanoparticles, and TRPV1, a temperature sensitivecation channel can be expressed in specific cells using specificpromoters. Second, cells are “decorated” by deliveringstreptavidin-coated iron oxide nanoparticles into the region where theBAP is expressed. The high affinity of streptavidin and biotin resultsin the cells being coated with the metallic/metal oxide nanoparticles.Studies in vitro have confirmed this to be the case. Third, the culturedcells, or an animal, are exposed to a RF field of defined strength at anintensity that will increase the local temperature of the nanoparticledecorated cells, activate the TRPV1 channel and thus triggering calciuminflux.

In some embodiments, the system can be modified such that only oneconstruct is used for both tethering of the nanoparticle and gating ofCa2+ entry. For example, the TRPV1 protein may be engineered as a fusionprotein capable of direct tethering of the nanoparticle. In anembodiment of the invention, the TRPV1 protein may be engineered as afusion protein containing any “tag” that does not interfere with thefunctioning of the channel. Such tags are well know to those of skill inthe art. As described in detail below, the TRPV1 protein can beexpressed as a fusion protein containing HIS tags. In such a case, thenanoparticles are coated with anti-HIS antibodies for targeting to thecell.

The present invention provides methods and compositions for studying therole of different cell types in a complex organism. The definitive testof cell function is to selectively turn on or off the activity of asingle cell type in a living animal and examine the effect onphysiological function. The present invention provides for the use ofnanoparticles to activate defined cell populations remotely withradiowaves. According to another embodiment, ferrous oxide coated withstreptavidin can be used to decorate cells, which express a biotinacceptor protein under the control of cell specific promoters. Thesesame cells are engineered to also express TRPV1, a single component,temperature-sensitive ion channel that can detect small changes intemperature within the physiological range and by conformational changeallow graded calcium entry. Exposing the metal coated cells to a definedelectromagnetic field increases the local temperature and activatesTRPV1 channels resulting in a Ca2+ current and cell activation. Data isprovided below that confirms the efficacy of this method both in vivoand in vitro. The technology can be used to modulate functions such ashormone release and neural activity. A means is also provided forcombinatorial activation of different cells using a modified TRPV1 andnanoparticles fabricated from other metals that can be excited atdifferent wavelengths. This tool can be used, for example, to examinethe roles of specific peripheral and CNS cell populations in energymetabolism.

The methods and compositions of the invention can be used to refine themethodology by decorating different cell types with distinct particlestuned to different wavelengths to activate ensembles of different cellpopulations in various combinations. Further, the ability of NICE tomodify hormone release to regulate glucose metabolism in diabeticanimals in vivo can be further refined. The methods and compositions canalso be used for stimulation of action potentials in electricallyexcitable cells to modify behavior and can be used to study the role ofspecific hypothalamic populations in (NPY and POMC) control of appetite.

Example 1 Radio-Wave Regulation of Plasma Glucose in Mice

The present invention provides methods and compositions to remotely andselectively switch on the activity of a single cell in a living organismand examine the effects on physiological function using nanoparticleinduced cell excitation (NICE). The technique targets temperaturesensitive calcium channels (TRPV1) to defined cells. These cells aredecorated with metal nanoparticles which are heated by an externalradiofrequency field. This in turn opens the TRP channel to stimulatecalcium influx. Calcium entry initiates downstream events such asdepolarization (neurons), hormone release (endocrine cells) or geneexpression.

The studies described herein demonstrate the efficacy of NICE atstimulating calcium influx, modulating hormone release and stimulatinggene expression both in vivo and vitro.

Material and Methods

Nanoparticles.

Iron oxide nanoparticles (10-50 nm diameter), functionalized with asurface carboxylic acid group, were purchased from Ocean Nanotech(Springdale, Ark.). The nanoparticles were conjugated to either mousemonoclonal anti-His antibody (AbD Serotec, Raleigh, N.C.) orstreptavidin (Jackson Immunoresearch Laboratories, West Grove, Pa.)using carbodiimide and N-hydroxysuccinimide activation technique asemployed by magnetic nanocrystal conjugation kit (Ocean Nanotech).Heating studies were performed using 1 ml of a bulk solution (1 mg/ml)of nanoparticles dispersed in water placed in an eppendorf inside thesolenoid and the temperature of the suspension was monitored using afiber optic system (Luxtron, Lambda photometrics, Herts, UK).

RF Magnetic-Field.

A 465 kHz sinusoidal signal was provided by a signal generator andapplied through an amplifier (both Ultraflex, Ronkonkoma, N.Y.) to a2-turn solenoid coil with a radius of 2.5 cm to produce a magnetic fieldstrength of 5 mT. Samples were placed within the solenoid. A 13.56 Mhzsinusoidal signal was provided by a signal generator (RFInstrumentation, PA) and applied through an amplifier (Comdel,Gloucester, M A) to a 2-turn solenoid coil with a radius of 2.5 cm).

Plasmids.

TRPV1 On pcDNA3.1) was a kind gift of Wolfgang Liedkte (Duke University,NC) and cloned into pEGFP-N1 (Clontech, Mountainview, Calif.). It wasmodified by PCR (Fast start PCR, Roche) to introduce His×6. Nuclearfactor of activated t-cells (NFAT) response elements and minimalpromoter were from pGL4.30[luc2P/NFAT-RE/Hygro (Promega, Madison, Wis.).Serum response element (SRE), cyclic AMP response element (CRE) and formmodified human insulin sequences were synthesized by Integrated DNAtechnologies (Coralville, Iowa). The calcium dependent responseelements—SRE, CRE and NFAT response elements were each used intriplicate. The use of these three response elements increased thelikelihood that at least one such mechanism would be active with highsensitivity in all of the cell types that we engineered. They were alsoused to achieve high specificity since each of these elements isreported to respond only to repeated or sustained changes inintracellular calcium. BAPTM, the transmembrane domain of plateletderived growth factor receptor fused to an extracellular biotin acceptorprotein, was a kind gift of Dr. B Tannous, Massachusetts GeneralHospital, MA). TRPV1^(His) and calcium responsive form insulin werecloned into MSCV-hygro and MSCV-puro plasmids (Clontech,) respectivelyfor retrovirus production using Phoenix packaging cells. Mouse ferritinheavy chain was obtained from ATTC (Manassas, Va.) in pCMV sport6 andmouse ferritin light chain 1 was obtained from Invitrogen (Carlsbad,Calif.) in pYX-Asc. These were cloned downstream of EF1alpha promoter inpCR2.1 with a flexible linker region to create ferritin lightchain-linker-heavy chain fusion protein. The fidelity of PCR productswas confirmed by DNA sequencing.

Cell Culture and In Vitro Studies.

Human embryonic kidney cells (HEK 293T) were cultured in Dulbecco'smodified eagle medium with 10% fetal bovine serum (Gibco, Carlsbad,Calif.) at 37° C. and 5% CO₂. PC12 cells were cultured in RPMI medium1640 with 10% horse serum and 5% fetal bovine serum (Gibco) at 37° C.and 5% CO₂. Phoenix ecotropic packaging cells (Stanford University) weregrown in Dulbecco's modified eagle medium with 10% fetal bovine serum(Gibco) at 37° C. and 5% CO₂.

Stable cell lines were produced by retroviral infection of PC 12 cellsusing the Phoenix system. Briefly, Phoenix eco cells (2×10⁶ cells per6-cm dish) were transfected with MSCV-puro or hygro plasmids asdescribed above. After 24 hours, the medium was replaced and the cellsplaced at 32° C. Medium was aspirated after a further 24 h and spun toremove cell debris. The Phoenix cell supernatant was added to PC12 cells(plated at 1×10⁶ cells per 6-cm dish) using a 1:2 dilution in RPMImedium/10% FBS with polybrene (4 μg/ml, Sigma-Aldrich, St Louis, Mo.).Cells were incubated at 32° C. for a further 24 h before replacing themedium with RPMI/10% FBS. Selection medium was added 48 h afterinfection.

Embryonic stem cells were electroporated with a calcium dependent humaninsulin plasmid and selected with puromycin for 3 weeks. Resistant cellswere identified for insulin insertion by Southern blot analysis beforeelectroporation with TRPV1His plasmid and selection with hygromycin for3 weeks. Three double resistant clones were screen by quantitative PCRfor expression of insulin and TRPV1 and by immunohistochemistry forexpression of TRPV1 and His tag. The ES clone with high expression byqPCR and IHC was used for RF studies. Cells were plated onto gelatincoated 12 mm cover glass (without feeder cells) for 96 hrs beforeassessing RF dependent release of calcium dependent human insulin.

For immunocytochemistry and RF studies, cells were cultured on 12-mmcover glass (Fisher Scientific, Pittsburgh, Pa.) coated with collagen(BD biosciences, Bedford, Mass.) and poly-D-lysine (Millipore,Billerica, Mass.). For EM studies, cells were cultured in 3 cm dishes.Cells were transfected 24 h after plating using lipofectamine 2000(Invitrogen, Carlsbad, Calif.). For temperature dependent studies, cellswere plated in 24-well plates coated with collagen and poly-D-lysine andtransfected 24 h later. Culture medium was replaced 18 h aftertransfection and holotransferrin (2 mg/ml, Sigma) was added to cellstransfected with ferritin. Cells were studied 72 h after transfection.

Temperature dependent release of calcium dependent human insulin: 24 hprior to the study, cells were placed in serum free medium at 32° C. toensure minimal activation of TRPV1 and calcium dependent pathways. Onthe day of study, cells were preincubated for 30 min in 500 μl phosphatebuffered saline (PBS) before incubation in 250 μl of calcium imagingbuffer (Caterina et al. 1997 Nature 389:816) at 32 or 44° C. for 1 h. Atthe end of I h, the supernatant was aspirated, spun to remove celldebris and frozen at −80° C. until assay. Studies were performed inquadruplicate on three occasions.

RF dependent release of calcium dependent human insulin: 24 h prior tothe study, cells were placed in serum free medium at 32° C. to ensureminimal activation of TRPV1 and calcium dependent pathways. On the dayof study, cells were preincubated for 30 min in 500 μl PBS or 500 μl offunctionalized iron oxide nanoparticles (1 mg/ml) resuspended in PBS.Cells were washed three times in PBS before incubation in 300 μl ofcalcium imaging buffer at room temperature (control) or in a RF field atroom temperature. The supernatant was removed at 15, 30, 45 or 60 mindepending on the study, spun to remove cell debris and frozen at −80° C.until assay. For gene expression analysis, cells from the supernatantand cover glass were lysed and the lysate stored at 80° C. until RNApurification. For apoptosis studies, the cells were incubated withfunctionalized nanoparticles at 1, 2, 4 or 8 mg/ml before RF treatmentand immunocytochemistry. For RF dependent time course from stable PC12cells, cells were left in serum containing medium prior to treatment tomore accurately replicate conditions in vivo before performing thestudies as described above. Medium was removed for assay at 15 and 30mins.

For studies examining the role of TRP channels in RF mediated proinsulinrelease, ruthenium red (100 μM) was added to the calcium imaging buffer.For studies to examine the role of calcineurin in the activation ofcalcium dependent insulin gene, Tacrolimus (100 nM, Tocris Bioscience,Minneapolis, Minn.) was added to the medium for 24 hours prior to RFtreatment. To examine if the effects of RF heating on iron oxidenanoparticles were confined to decorated cells, 3-cm dishes of HEK 293Tcells were transfected with either BAPTM or TRPV1 and calcium dependenthuman insulin. After 24 h, the two transfected cell populations weretrypsinized and mixed to form a co-culture with adjacent cellsexpressing BAPTM and TRPV1/human insulin.

Calcium Imaging.

Transfected cells were washed three times in PBS then loaded with Fluo-43 μM (Invitrogen) in the presence of sulfinpyrazone 500 μM (Sigma) for60 min at room temperature. Cells were washed again in PBS thenincubated for 30 min either with functionalized nanoparticles withsulfinpyrazone or in sulfinpyrazone in PBS. Cells were washed and thenimaged in calcium imaging buffer. Imaging was performed using aDeltavision personal DV imaging system (Applied Precision, Issawaq,Wash.) equipped with a custom-made ceramic lens. Cells were imagedbefore and during RF treatment or before and after treatment with 200 μM2 aminoethoxydiphenyl borate (2-APB).

Immunocytochemistry, Immunohistochemistry and Electron Microscopy.

Immunocytochemistry (ICC) was used to detect and quantify apoptoticcells following RF field treatment. Two assays were employed, TUNELassay visualized using Apoptag Fluorescein direct in situ apoptosisdetection kit (Millipore) and ICC for activated caspase-3 (Promega).After RF treatment, cells were washed twice in PBS and then fixed for 15min in 2% paraformaldehyde (Electron Microscopy Services, Hatfield,Pa.). Cells were then stained according to the manufacturers' protocols.Expression of NFAT1 was also examined using ICC. Control or RF treatedtransfected cells were washed and fixed as above then incubated in for 1h in blocking buffer (3% BSA (Sigma) and 2% goat serum (Sigma) in PBSwith 0.1% Triton-X (Sigma)). Following blocking cells were incubated inprimary antibody, rabbit anti-NFAT1 (Cell signaling), 1:50, diluted inblocking buffer overnight at 4 degrees. Cells were then washed threetimes in PBS before incubation in secondary antibody (goat anti-rabbit488 1:1000) diluted in blocking buffer for 1 h. The cells were thenwashed a further three times in PBS before inverting and mounting usingFluoromount with DAPI (Southern Biotech, Birmingham, Ala.).

ICC was also used to examine expression of ferritin fusion. Cells werewashed twice with PBS, fixed and blocked as above. Cells were incubatedin primary antibody, rabbit anti-ferritin light chain (Dako,Carpinteria, Calif.), 1:1000, diluted in blocking buffer for 1 h. Cellswere washed three times in PBS before incubation in secondary antibody(goat anti-rabbit 488 1:1000) diluted in blocking buffer for 1 h. Thecells were washed a further three times in PBS before inverting andmounting using Fluoromount (Southern Biotech, Birmingham, Ala.).

Immunohistochemistry (IHC) was used to confirm expression of TRPV1 andHis and quantify apoptotic cells in tumors. Tumors were fixed in 10%formalin (Sigma) at 4° C. overnight then placed in 30% sucrose in PBS at4° C. for a further 24 h. Tissue was embedded in OCT and frozen before20 μm cryosections were cut and placed directly on glass slides. Slideswere placed at 55° C. for 1 h then stored at −80° C. before stainingApoptag Fluorescein direct in situ apoptosis detection and IHC foractivated caspase-3 were performed according to the manufacturers'instructions. Staining for TRPV1 and His was performed as follows.Slides were washed three times with PBS then incubated in blockingbuffer for 2 h followed by overnight incubation at 4° C. with primaryantibody diluted in blocking buffer (rabbit anti-TRPV1 1:500, mouseanti-His 1:1000 (Sigma)). Slides were washed 3-times in PBS and thenincubated overnight at 4° C. with secondary antibody diluted in blockingbuffer (goat anti-rabbit 488 and goat anti-mouse 594 both at 1:1000).Slides were washed 3-times in PBS before applying Fluoromount and acoverglass (Fisher Scientific).

Images were acquired using a Zeiss Axioplane microscope and captureddigitally with separate band-pass filters using the multichannel moduleof the AxioVision Zeiss software. Additional images were acquired usingconfocal microscopy (LSM 510 laser scanning confocal microscope; CarlZeiss MicroImaging, Inc.). Quantification of TUNEL and active caspase-3immunostaining was performed by an investigator blinded to the treatmentgroup.

Electron microscopy was used to quantify nanoparticle binding to thecell membrane and to image ferritin in transfected cells. Cells werefixed in 2% paraformaldehyde/2.5% glutaraldehyde/0.1M cacodylate buffer,pH 7.4, for 15 minutes before pelleting and further fixation for 1 h.Cells were then treated with 1% osmium tetroxide (1 h, on ice) and 0.5%uranyl acetate (1 h) before dehydration with graded ethanol andtreatment with propylene oxide (2×15 min). The cells were infiltratedwith 50% EPON epoxy resin (Miller-Stephenson, Sylmar, Calif.) and 50%propylene oxide overnight then 100% EPON (2×2 h) before curing at 60° C.for 2 days. Blocks were cut with a diamond knife on a Leica UltracutE(Buffalo Grove, Ill.) and ultra-thin (˜70 nm) sections were collected onuncoated 200 mesh grids and stained with uranium and lead. Grids wereviewed with a Tecnai SpiritBT Transmission Electron Microscope (FEI,Hillsboro, Oreg.) at 80 KV and pictures were taken with Gatan 895ULTRASCAN Digital Camera (Pleasanton, Calif.).

ImmunoEM was used to confirm binding of nanoparticles to TRPV1. HEK 293Tcells were seeded on Aclar (Ted Pella Inc, Redding, Calif.) in 24 wellplates and transfected with TRPV1 His 24 hours later. 72 hours aftertransfection, cells were washed twice with PBS and incubated withanti-His coated iron oxide nanoparticles for 30 mins. Following twofurther PBS washes, cells were fixed in a fixative containing fresh 4.0%paraformaldehyde and 0.1 M sodium cacodylate buffer (pH 7.4) forovernight. Cells were processed for immunoelectron microscopy: afterincubating with a blocking solution containing 3% BSA and 0.05% saponinin sodium cacodylate buffer for 20 min, the primary antibody was appliedand incubated for overnight at 4° C.; in the following day, afterextensive wash secondary antibody conjugated with nanogold (Nanoprobes,Inc. Yaphank, N.Y.) (1:50) was applied and incubated for 2 hours;subsequently, with series of wash, silver enhancement was conducted byusing silver enhancement kit (Nanoprobes, Inc. Yaphank, N.Y.). Cellswere re-fixed with 2.5% glutaraldehyde in the cacodylate buffer, lightlypostfixed with 1% osmium tetra-oxide (10 min), dehydrated by a gradedseries of ethanol, infiltrated with EMBed812 resin and embedded in thesame resin. Ultra-thin sections were cut and examined in the electronmicroscope (100CX JEOL, Tokyo, Japan) with the digital imaging system(XR41-C, Advantage Microscopy Technology Corp, Danver, Mass.). Controlexperiment was done by following the same procedure except for the stepof omitting primary antibody and applying the blocking solution,instead.

Animals and In Vivo Studies.

Male athymic NCr-nu/nu mice (NCI-Frederick, 6 weeks old), an outbredstrain, were used and housed under controlled light conditions (12 hlight/12 h dark) and temperature (22° C.), single-caged, and fed adlibitum on standard mouse chow. Animal care and experimental procedureswere performed with the approval of the Animal Care and Use Committee ofRockefeller University (protocol 11421) under established guidelines.

To generate subcutaneous tumors, 5×10⁶ PC12 cells stably expressingTRPV1^(His) and calcium dependent human insulin or calcium dependenthuman insulin alone were resuspended in a 1:1 mixture of PBS andgrowth-factor reduced Matrigel (BD biosciences) and injected into theflank of anesthetized mice bilaterally. Radiofrequency studies wereperformed 4 weeks later. Blood glucose was monitored weekly. Mice werefasted overnight before all studies.

Study 1:

Mice with TRPV1^(His)/insulin xenografts (n=7) were anesthetized withinhaled isoflurane. PBS or iron oxide nanoparticles functionalized withanti-His antibody were injected into the tumor (5×10 μl). After 30 min,mice were treated with an RF magnetic field for 30 min by placing in asolenoid connected to the RF generator. Tail vein samples were taken at−30 and 0 min before RF magnetic field treatment and at 10, 20, 30, 45,60, 90 and 120 min after the onset of RF treatment. Retro-orbital bloodwas taken using EDTA coated capillary tubes at −30 and 30 min for plasmainsulin measurement. As the mice are outbred and had highinter-individual variability in blood glucose, the studies wereperformed with a cross-over design with each mouse receiving first PBSand then one week later receiving nanoparticle injections. Nanoparticleinjections could not be performed first because the particles remainwithin the tissue for prolonged periods (Giustini et al., 2011Nanotechnology 22:345101).

Study 2:

Mice with TRPV1^(His)/insulin xenografts (n=6/group) were anesthetizedand were randomized to receive either functionalized iron oxidenanoparticles or PBS. An identical treatment protocol to study 1 wasused and intratumoral temperature and core body temperature weremonitored using a fiber optic temperature monitoring system. Thermalimaging using FLIR infrared camera SC325 (North Billerica, Mass.) wasperformed on a subset of mice. After 120 min, the mice were sacrificedand the tumors removed. Each tumor was divided in two and one half snapfrozen in liquid nitrogen for RNA extraction and the one half placed in10% formalin for immunohistochemistry.

Study 3:

Mice injected with PC12 cells stably expressing calcium dependent humaninsulin (n=8) were used and study 1 repeated.

Study 4:

Mice injected with PC12 cells expressing calcium dependent human insulinwere used (n=6/group) and study 2 repeated.

Study 5:

Mice injected with PC12 cells expressing TRPV1^(His) and calciumdependent human insulin were used (n=6/group) and study 1 was repeatedusing nanoparticles which had not been conjugated to anti-His antibody.

Study 6:

Mice injected with PC12 cells expressing TRPV1^(His) and calciumdependent human insulin were used (n=7/group). Study 1 was repeated withmice injected initially with PBS and treated with RF. Blood glucose andinsulin were measured as described above. One week later, mice wereinjected with anti-His conjugated nanoparticles and treated with RF.Blood glucose was monitored as above. After a further week, micereceived a second injection of anti-His conjugated nanoparticles, weretreated with RF and blood glucose and insulin were measured.

Assays.

Proinsulin was measured in cell supernatants by ELISA (Alpco, Salem,N.H.) according to manufacturer's protocol. Blood glucose was determinedusing a Breeze 2 glucometer (Bayer; Leverkusen, Germany). Blood was spunfor 10 min and plasma was collected. Plasma levels of human insulin,produced by xenografts, were determined in mouse plasma by humanspecific ELISA (Alpco).

Real-Time PCR.

Total RNA was isolated by homogenizing tissue in TRIzol reagent(Invitrogen) or cells in buffer RLT and purifying the RNA using QIAGENRNA prep kit. Complimentary DNA was synthesized using QIAGEN omniscriptRT kit. Real-time PCR was performed using the TaqMan system (AppliedBiosystems; Foster City, Calif.) according to the manufacturer'sprotocol.

Statistics.

All data were analyzed for statistical significance using the Student'st test. P values are as indicated.

Nanoparticle Characterization.

Iron oxide nanoparticles purchased from Ocean Nanotech (Catalog #SHP-20-50) were further characterized. The particles were coated witholeic acid and a proprietary polymeric coating, which iscarboxy-terminated for further functionalization. The particles werespecified to be 20 nm in diameter with a standard deviation of <5%. Thiswas confirmed in the current work; TEM analysis of the size distributionyielded an average diameter of 19.8±2.7 nm (FIG. 3B). The particles wereestimated to have a 4 nm oleic acid/polymeric coating in addition to thenanoparticle diameter observed in TEM. X-ray photoelectron spectroscopy(XPS) allowed identification of all elements within ˜5 nm of thenanoparticle surface, as well as a more precise investigation of thenanoparticle surface chemistry and iron oxide content. XPScharacterization (FIG. 3C) of the IONP indicated only the presence ofiron, oxygen and carbon as would be expected from the polymeric coating.The shoulder in the C1s peak is consistent with the presence of carboxylgroups, as indicated in FIG. 3D. Moreover, the structure of the ironoxide particles was indicated by Ocean Nanotech to be magnetite (Fe₃O₄)for this particle size. However, magnetite and maghemite (γ-Fe₂O₃) isdifficult to distinguish using x-ray diffraction (XRD) due tosimilarities in crystal structure. Indeed, comparison with JCPDS files#39-1346 (maghemite) and #75-0033 (magnetite) indicates that the currentiron oxide particles are likely a mixture of both crystallographicstructures.

Studies were also performed to extend the analysis of the heatingcharacteristics of the nanoparticles at different frequencies. A limitednumber of frequencies are suitable for medical use in addition to the465 kHz used previously. The heating of particles using a frequency of13.56 Mhz which is the lowest of the remaining available frequencies andtherefore the least likely to result in non-specific adsorption was alsotested. The power level chosen was the maximum level that did not resultin significant heating of water (200 W) in the absence of nanoparticles.At 465 kHz, observed a rapid increase in temperature for a suspension of20 nm iron oxide nanoparticles with a temperature increase of 17° C.that reached a plateau at 300 s. A smaller rise in temperature (6° C.)was seen with 25 nm particles with only minimal heating for larger sizesof iron oxide nanoparticle. In contrast, at 13.56 MHz, there wassignificant heating for a broader range of particle sizes (10-25 nm)with greatest heating observed for 15 nm particles (11° C.) and verylittle heating of 20 nm particles, with only a maximum temperature riseof 2° C. However, the heating rate for 15 nm particles was lower thanthat for 20 nm particles at 465 kHz and did not plateau over the testperiod. The heating of 25 nm particles was similar at both frequencies.

Nanoparticle Heating.

Temperature decay from the iron oxide nanoparticle (IONP) surface hasbeen explored (Rast and Harrison, 2010 Nanotech Conference and Expo2010, Anaheim, Calif., June 21-24, p. 910-914; Hergt et al., 2010 IEEETrans. Mag. 34:3745; Wilson O. et al., 2002 Phys. Rev. B 66:224301;Jordan et al., 1993 Int. J. Hyperthermia 25:499) and is complicated byfactors such as the heat capacity of the surrounding medium, thenanoparticle ligand coating, and diffusion away from the nanoparticlesurface. The change in temperature in the vicinity of the nanoparticledecreases as the inverse of the radius, and may be approximated by theconductive heat transfer equation (Fourier's Law)(Rabin, 2002 Int. J.Hyperthermia 18:194):

${\Delta\; T} = {{\frac{V_{np}Q}{4\pi\; k}\frac{1}{r}} = \frac{q\;\rho\; D^{2}}{12k}}$

where V_(np) is the nanoparticle volume, Q is the total dissipated heat,r is the nanoparticle radius, q is the heating rate (W g⁻¹), ρ is thenanoparticle density, D is the diameter of the heated volume, and k isthe thermal conductivity of water (0.64 W m⁻¹° C.⁻¹). Though it has beencalculated that heating sufficient to trigger channel activation cannotbe achieved on the single nanoparticle scale (AT˜10⁻⁵° C./particle), ithas been shown that accumulating a significant number of iron oxidenanoparticles in a small volume may raise the temperature sufficientlyto have an effect. In a separate study, thermoresponsive polymercoatings on iron oxide nanoparticles have demonstrated increased drugrelease in radiofrequency fields, indicating that heating at thenanoparticle surface affected the structure of the thermopolymer anddrug release profile. Both calculations and experiments in theliterature have confirmed that ˜1.2 mg of IONP are required to raise thetemperature of a 1 cm³ region by 5° C. (Derfus et al., 2007 Adv. Mater.19:3932). Based on this published literature and assuming a cell volumeof 9.05×10⁻¹⁰ ml, we would require 1.1×10⁻⁹ mg/cell (ie 1.1 pg/cell) togive a nanoparticle density of 1.2 mg/ml to cause a local 5° C. increasein temperature. From EM images, we observe an average of 2×10⁵nanoparticles/cm. Assuming the particles are evenly distributed, thiswould give 4×10¹⁰ nanoparticles/cm². For a 20 μm cell, the surface areais 1.25×10⁻⁵ cm² (assuming a sphere) and therefore the calculated numberof nanoparticles decorating the cell is 5×10⁵ nanoparticles. The densityof Fe₃O₄ is 5.24 g/cm³, and each nanoparticle is 20 nm in diameter,giving a nanoparticle weight of 1.75×10⁻¹⁶ g. Therefore, to achieve 1.1picograms of nanoparticles per cell one would need at least 6.26×10³nanoparticles. Thus it is calculated that an almost 100 fold greaternanoparticle decoration than that required to achieve a 5° C. localincrease in temperature has been obtained.

Results

A system has been developed that allows remote activation of proteinproduction by engineered cells in vitro and in vivo. The method(Figure. 1) uses iron oxide nanoparticles (FeNPs) that are coated withantibodies against His (anti-His) and that bind a modified TRPV1 channelwith an extracellular His×6 epitope tag (TRPV1^(His)). As disclosedherein RF treatment, local heating of bound anti-His FeNPs activates thetemperature-sensitive TRPV1, resulting in a calcium current to activatea Ca²⁺-sensitive promoter placed upstream of a modified human insulinreporter gene.

FeNPs was used for the following reasons: They heat at 465 kHz, arelatively low frequency that minimizes tissue heating; particles of 20nM or less diffuse in the extracellular space (Wang et al., 2008 ExpertOpin. Biol. Ther. 8:1063; Deflaco et al. 2001 Science 291:2608; Thorneet al., 2006 Proc. Natl. Acd. Sci USA 103:5567); and these particles canbe derivatized with antibodies. At 465 kHz (5 mT), substantial heatingwas observed for 20- and 25-nm FeNP suspensions (FIG. 2). A 20-nm FeNPsuspension had an initial heating rate of 0.15° C./s and a specificabsorption rate (SAR) of 0.63 W/g, whereas the SAR of water at thisfield frequency and strength was less than 0.004 W/g. As shown byelectron microscopy, a His-tag insertion into the first extracellularloop of TRPV1 provided a site for significant and specific FeNP binding(FIG. 3) with direct heat transfer to the adjacent channel (FIG. 4).Human embryonic kidney (HEK) 293T cells expressing TRPV1^(His) anddecorated with 20 nM FeNPs conjugated to anti-His showed a significantincrease in intracellular Ca²⁺ after 10 s of RF exposure (FIG. 3D).

Calcium entry was next used to induce gene expression via a novelsynthetic 5′ regulatory region composed of three Ca²⁺ response elementsin cis: serum response element (SRE), cyclic adenosine monophosphateresponse element (CRE), and nuclear factor of activated T cell responseelement (NFAT RE) (Hardingham et al. 1999 Microsc. Res. Tech. 46:348;Rao 2009 Nat. Immunol. 10:3) with a minimal promoter. This was placedupstream of a modified human proinsulin gene with furin cleavage sitesreplacing beta-cell-specific convertase cleavage sites to allow insulinprocessing in non-beta cells (Shirin et al., 2001 Gene Ther. 8:1480)(FIG. 5A).

HEK 293T cells expressing the Ca²⁺-dependent human insulin construct andTRPV1^(His) were incubated with functionalized FeNPs. RF treatment ofthe FeNP-decorated cells resulted in a significant increase inproinsulin release (RF-treated 671±235% (SEM) basal versus 100±13.9% forcontrols, P<0.02) and insulin gene expression (RF-treated 2.20±0.53insulin gene expression relative to basal versus 1.0±0.18 for controls,P<0.05). These were blocked by the TRP channel inhibitor, ruthenium red(FIG. 6A). There was a trend toward an increase in proinsulin releaseafter 15 min of RF treatment (likely through release of a small pool ofpreformed insulin-containing vesicles), with significant proinsulinrelease at 1 hour when insulin gene expression had also significantlyincreased (FIG. 6B). Control studies confirmed that proinsulin releaserequired all system components (i.e., TRPV1, nanoparticles, and RFmagnetic field) and that RF-dependent insulin secretion was confined toFeNP-decorated cells (FIG. 5B-C). RF treatment induced NFATtranslocation into the nucleus, and RF-dependent proinsulin release wasblocked by a calcineurin inhibitor, tacrolimus (FIG. 5D-E). RF treatmentof cells incubated with FeNPs (1 to 8 mg/ml) did not induce apoptosis asassessed by immunohistochemistry for active caspase 3 (Tewaari et al.,1995 Cell 81:801)) and terminal deoxynucleotidyl transferase-mediateddeoxyuridine triphosphate nick end labeling (TUNEL) (Gavrieli et al 1992J. Cell Biol. 119:493) (FIG. 5F-G). It was also shown that RF couldstimulate proinsulin release from mouse embryonic stem cells expressingTRPV1^(His) and the Cat*-dependent human insulin construct (FIG. 7).

Next, a comparison was made of the single-component system to amulticomponent system previously reported (Huang et al., 2010 Nat.Nanotechnol. 5:602) and composed of (i) a membrane-tethered biotinacceptor protein, (ii) a bacterial biotin ligase to biotinylate thisprotein to enable binding of (iii) streptavidin-coated nanoparticles,and (iv) a wild-type TRPV1 as the effector. Although this system inducedCa²⁺ entry in vitro and activated endogenous temperature sensing ofCaenorhabditis elegans neurons, the complete system was not tested invivo because the exogenous TRPV1 channel was not used in this priorstudy. It was found that the single-component system had densernanoparticle binding, faster calcium entry, and a trend toward morerobust proinsulin release (FIG. 8A-G). These attributes led to thetesting of the single-component TRPV1^(His) system in vivo in mice byusing xenografts of engineered neuroendocrine PC12 cells, which robustlysecrete peptides via the regulated pathway for protein secretion.

Exposure of a PC-12 cell line stably expressing TRPV1^(His) and thecalcium-dependent human insulin construct (PC12-TRPV1^(His)-Ins) to RFafter FeNP application significantly increased proinsulin release andinsulin gene expression in vitro (FIG. 9A-D). The PC12-TRPV1^(His)-Inscells were injected subcutaneously into the flank of nude mice to formtumors (FIG. 9F). There was no change in plasma glucose with tumorgrowth (FIG. 9E). Phosphate-buffered saline (PBS) or FeNP (50-μl totalvolume, nanoparticle concentration of 8 mg/ml) were injected into thetumors of fasted mice, and blood glucose and plasma insulin weremeasured before, during, and after RF application (see FIG. 11A forprotocol). RF treatment resulted in a significant decrease in bloodglucose in nanoparticle-treated PC12-TRPV1^(His)-Ins tumor mice (FIG.10A) (The change in blood glucose at 30 min for FeNP-treated was−53.6±8.90 mg/dl, versus PBS-treated, −11.0±9.72 mg/dl; P<0.005. At 45min, the change in blood glucose for FeNP-treated mice was −60.9±11.6mg/dl versus PBS-treated −9.74±8.52 mg/dl; P<0.005. At 60 min, thechange in blood glucose for FeNP-treated mice was −55.1±13.2 mg/dlversus PBS-treated, −6.24±15.3 mg/dl; P<0.0001). There was also a highlysignificant difference in the cumulative change in blood glucose (areaunder the curve, AUC) between PBS-treated and FeNP-treatedPC12-TRPV1^(His)-Ins tumor mice over the course of the study (FIG. 10B)[AUC (0 to 120 min) for PBS-treated was 272±692 mg/dl min versusFeNP-treated, −2695±858.3 mg/dl min; P<0.002]. Plasma insulin wassignificantly increased after RF treatment in FeNP-treated but notPBS-injected mice (FIG. 10C) [plasma insulin for FeNP-treated (−30 min)was 2.26±0.76 μI-U/ml (where 1 μI-U/ml=0.006 pmol/l) versus FeNP-treated(30 min), 3.25±0.64 μI-U/ml; P<0.05. PBS-treated (−30 min) was 1.83±0.38μI-U/ml versus PBS-treated (30 min), 1.75±0.36 μI-U/ml]. Lastly, insulingene expression was significantly increased in RF-treated, FeNP-injectedtumors (FIG. 10D) (relative insulin gene expression in FeNP-treated, noRF samples was 1.0±0.2 versus FeNP-treated with RF, 2.0±0.3, P<0.05)without increasing c-fos expression (FIG. 11B). Core temperature did notchange significantly with RF exposure, and the intratumoral temperatures(23° to 31° C.) remained well below the 42° C. threshold of TRPV1,indicating that cell activation is due to localized, cellsurface-specific gating of TRPV1. There was no difference in apoptosisbetween FeNP-injected tumors in the presence or absence of the RFmagnetic field (FIG. 11C-D). A significant decrease in blood glucose wasalso seen with serial FeNP injection and RF treatment (FIG. 11E-F). Toconfirm that the effects on blood glucose were not due to nonspecificinsulin release via thermal effects of nanoparticles on the tumor, thein vivo study was repeated, first in mice injected with PC12 cellsexpressing the Ca²⁺-dependent human insulin construct but notTRPV1^(His) and then in mice with tumors expressing TRPV1^(His) andinsulin but injected with nanoparticles without anti-His conjugation.There was no significant effect on blood glucose in either study afterPBS or FeNP injection and RF treatment despite similar intratumoraltemperatures (FIG. 12A-D).

The above approach requires direct application of nanoparticles to TRPV1expressing cells by incubation (in vitro) or injection (in vivo). Analternative is to engineer cells to synthesize nanoparticlesintracellularly. For this purpose, the iron storage protein ferritin waschosen, which forms a naturally occurring iron nanoparticle (Farrant,1954 Biochim. Biophys. Aca 13:569; Sana et al., 2010 Biointerphases5:FA48) (FIG. 13A, FIG. 14). Ferritin has been stably overexpressed inmice for over 2 years without evident pathology (Ziv et al., 2010 NMRBiomed 23:523). A fusion peptide of ferritin light chain, flexiblelinker region, and ferritin heavy chain fixes the ratio of light toheavy chains and increases iron binding (Iordanova et al., 2010 J. Biol.Inorg. Chem. 15:957). Transfecting cells with ferritin fusion proteinresulted in 12.6±2.86 ferritin particles per 0.2 μm² with an averagedistance to the cell membrane of 60.3±2.85 nm (FIG. 14B). RF treatmentof cells expressing the ferritin fusion protein, TRPV1, andCa2+-dependent human insulin significantly increased proinsulin release(RF treated, 457±103% basal versus control, 100±14.9% basal; P<0.005)and insulin gene expression (relative insulin gene expression for RFtreated, 1.58±0.19 versus control, 1.0±0.17; P<0.05) (FIG. 13B).Intracellularly generated FeNPs were roughly two-thirds as effective instimulating proinsulin release as exogenous FeNP. In the absence ofTRPV1, insulin secretion and gene expression were unchanged inferritin-expressing cells. Thus, ferritin expression may provide agenetically encoded source of nanoparticles for RF-mediated cellactivation.

As disclosed herein both externally applied and endogenously synthesizednanoparticles can be heated by radio waves to remotely activate insulingene expression and secretion. RF-mediated cell activation does notrequire a permanent implant, and the cells to be activated can belocalized (when using exogenous nanoparticles) or dispersed (by usinggenetically encoded nanoparticles). Genetically encoded ferritinnanoparticles may also provide a continuous source of nanoparticles forcell activation.

The use of an epitope-tagged TRPV1 with antibody-coated nanoparticlesresulted in high nanoparticle density in proximity to the channel andcould gate calcium in response to power levels of a 465-kHz RF fieldthat are within Food and Drug Administration guidelines (Halperin etal., 2008 Proceedings of the 2008 IEEE Symposium on Security and PrivacyOakland Calif. May 18-21 2008 p. 129-142). The use of a single constructfor particle binding and calcium entry also simplifies DNA deliveryusing viral vectors or other approaches (Nathwani et al., 2011 N. Engl.J. Med. 365:2357). Additionally, an epitope-tagged channel offers thechoice of activating distinct cell populations in the same organism withdifferent RFs to selectively and independently heat nanoparticles boundto cell specific tags. For endogenous particles, mutations of ferritinthat alter the metal it encapsulates could enable combinatorial cellactivation (Butts et al., 2008 Biochemistry 47:12729).

A noninvasive, nonpharmacological means has been developed for cellstimulation and validated it in vitro and in vivo. This system providesa useful tool for basic research and represents an initial step towardnoninvasive regulation of protein production for possible therapeuticpurposes. This approach could be used to treat protein deficiencies byproviding regulated expression of proteins that are difficult tosynthesize or to deliver [such as central nervous system (CNS)replacement of hexosaminidase A for Tay-Sachs] or to allow CNS deliveryof recombinant antibodies to treat brain metastases. This approach couldalso enable the activation of other Ca²⁺-dependent processes, such asmuscle contraction or firing of action potentials.

Example 2 Nanoparticle Induced Cell Excitation

Effects of NICE In Vivo.

Data is provided herein which shows the effectiveness of NICE in vivo.For these studies the effect of NICE on insulin release was examined inimplanted cells. Stably transfected PC12 cells expressing TRPV1, ananoparticle tether and insulin driven by a calcium dependent promoter(NFAT-insulin) were implanted into nude mice to form subcutaneoustumors. In a cross-over design, either vehicle (PBS, 5×10 ul) orfunctionalized nanoparticles (20 nm, 5×10 ul) were injected into eachtumor (n=8 mice). Thirty minutes after injection, mice were exposed to aradiofrequency field (RF) for a period of 30 minutes. Blood glucose andinsulin were monitored and the tumors harvested at the end of the studyperiod. This protocol is described visually in FIG. 20A. Mice receivingnanoparticle injection and RF showed a significant reduction in bloodglucose, a significant increase in circulating insulin and a significantincrease in tumor expression of insulin (FIG. 20B-E). There was nodifference in apoptosis between groups based on immunohistochemicalassessment of TUNEL or activated caspase-3.

The results demonstrate the usefulness of NICE as a tool fornon-invasive stimulation of cell function in vivo. Additional studiescan be performed to examine the effect of stimulating endogenous cellpopulations using transgenic mice expressing a nanoparticle tether andTRPV1, with or without calcium dependent gene expression. It is alsopossible to examine the effects of stimulating cell populations derivedfrom individual-specific induced pluripotent stem cells (iPSC) injectedinto immune-competent mice. Viral free iPSC populations can bereprogrammed to a wide variety of tissues and also modified to expressNICE components. These cell populations would allow the non-invasivestudy of specific stimulation of defined cell types and/or defined geneswithin these cells providing a valuable tool in the investigation ofcell function particularly as it pertains to complex behaviors.

Intracellular Nanoparticle Synthesis.

In its present form, NICE technology requires external nanoparticleapplication by incubation (in vitro) or injection (in vivo). Forperipheral tissues, this may be achieved by intravenous administrationbut modulation of neuronal activity requires intracerebraladministration. This is advantageous in some situations wherestimulation of an anatomically defined subpopulation of a dispersedneural group is needed. However, a variation of the existing NICEtechnology with intracellular nanoparticle synthesis using chimericferritin proteins targeted to an intracellularly tagged TRPV1 channel isbeing studied.

The iron storage protein, ferritin, is a complex of heavy and lightchains that is capable of binding 4500 ferric ions or picograms of ironper cell. This iron binding capacity has allowed ferritin to be utilizedas an MRI contrast agent in many studies. Two chimeric ferritin peptideshave been developed, one fused at the N-terminal to Egfp and one fusedto a highly stable, high affinity (subnanomolar) camelid anti-gfpantibody known as a nanobody (FIG. 21A). These adaptations allowadjacent ferritin complexes to bind to form ferritin aggregates and EMimages show these are of the order of 40-50 nm diameter (FIG. 21B). Inaddition, the temperature dependent calcium channel, TPRV1, is modifiedby attaching the nanobody sequence to its intracellular N-terminal whichthen tethers the ferritin aggregate. This brings the heating component(ferritin bound iron) and the effector component (TRPV1) of NICEtogether at the cell surface.

There are 4 forms of the ferritin complex that have been tested—ferritinlight chain-linker-heavy chain (ferritin complex) expressing in thecytoplasm, ferritin complex expressed at the cell membrane, ferritincomplex with egfp fusion that binds to TRPV1 with anti-gfp camelidantibody fusion and the system described above. FIG. 15 demonstratesproinsulin release from 293T cells transfected with calcium dependentinsulin and either TRPV1/myristoylated ferritin, camelid anti-GFP fusedto TRPV1/EGFP ferritin or camelid anti-GFP fused to TRPV1/EGFPferritin/camelid anti-GFP fused to ferritin in response to RF treatment.

Example 3 NICE: Iron Oxide Nanoparticles

Iron oxide nanoparticles, functionalized with streptavidin to decoratethe cells have been used for several reasons. Firstly, thestreptavidin-biotin complex is the highest affinity non-covalent bondknown and directs the nanoparticles stably to the cell surface.Secondly, the heat generated by nanoparticles in an RF field depends onboth Brownian motion and Neel fluctuation, the internal rotation of themagnetic moment. When nanoparticles are bound to the cell surface, theproportion of heat generated by Brownian motion is significantlydecreased. In contrast to other particles, such as cobalt ferritenanoparticles, heat generated by iron oxide particles is primarilythrough Neel fluctuation rather than Brownian motion (Fortin et al.,2007 J. Am. Chem Soc. 129:2628-2635). Thirdly, functionalized iron oxidenanoparticles are readily available in a number of sizes allowing theheating response to be tuned through field strength and frequency, andparticle size. The size of particles that diffuse freely in a number ofin vitro and in vivo settings has been defined and 20-30 nm beads havebeen found to be optimal.

Using an alternating magnetic field (465 kHz, 110 kA/m) provided by acommercial induction generator (Ultraflex Power Technologies) and acustom-made, water cooled, induction coil (4.5 cm diameter), it has beendemonstrated that the temperature of a suspension of iron oxidenanoparticles increases when it is exposed to the appropriate RFfrequency. While much lower temperature increases are necessary for NICEin vitro or in vivo, an increase of up to 17° C. can be achieved when asuspension of 20 nm iron oxide particles (1 mg/ml) are exposed to a 465kHz, 110 kA/m field (FIG. 22). It has been noted that a 5° C. rise innanoparticle temperature is sufficient to open TRPV1 channels and can bereached in approximately 30 s with no increase in water temperature.

Iron oxide nanoparticles coated with streptavidin were used to decoratespecific cell populations by expression of a tether in the form of abiotin acceptor protein (BAP) fused to the transmembrane domain of PDGFRand tagged with hemagglutinin (HA) (Tannous et al., 2006 Nat Methods3:391-396) The biotin acceptor protein is modified by endogenous biotinligase. The affinity of streptavidin and biotin is 10-15M and this highaffinity has been shown by us to lead to specific binding of thenanoparticles only to cells that express the BAP tether. It has beenshown that nanoparticles can decorate transfected HEK293t cells thatexpress an HA tagged BAPTM fusion protein (FIG. 23). Electron microscopyconfirms that streptavidin-coated ferrous oxide nanoparticles bind tothe cell membrane and quantification reveals that this is significantlygreater on cells transfected with the BAPTM fusion protein versus cellsnot expressing this construct. In addition, immunocytochemistrydemonstrates significant co-localization of TRPV1 with HA (BAP)immunoreactivity, TRPV1 with biotin immunoreactivity, and TRPV1 withAlexa 594-streptavidin binding. These results confirm that constructscan drive co-expression of TRPV1 and the BAPTM fusion protein in thesame cells (FIG. 23).

With NICE, the local temperature change achieved by heatingnanoparticle-coated cells is transduced into calcium entry by targetedexpression of TRPV1 channel. This is a single component channel thatdetects small changes in temperature just above the physiological range(>42° C.) as a result of a conformational change allowing calcium entry(Caterina et al., Nature 389:816-824). The response is proportional tothe temperature change and relatively rapid, thus transducingtemperature variations induced by the heated nanoparticles into a gradedcalcium current with attendant cell activation. Repeated heating alsopotentiates calcium entry, i.e., there is no desensitization. Generally,ion channels such as TRPV1 offer many advantages for modifying cellactivity, including as described above, their fast temporal resolutionand the ability to target their expression genetically without thetissue damage often seen with direct electrical stimulation. In thisrespect the activation of a temperature sensitive channel provides thesame advantages as the reported light activated cation channel butwithout the need for an indwelling catheter, and with the potential, byusing different nanoparticles, to effect activation of different celltypes in response to different RF frequencies to modulate multiple cellsin the same local region.

It has been confirmed that NICE can induce calcium entry in response toradio waves using streptavidin-coated iron oxide nanoparticles,expression of a cell surface biotin acceptor protein and the temperaturesensitive TRPV1 channel in transfected HEK293t cells both with calciumimaging and a calcium sensitive NFAT-luciferase reporter construct asthe readout (FIG. 24). The calcium response can be modulated both by thepower of the RF field and by the exposure time. These findings confirmthe feasibility of the method in vitro. The methodology in vivo invertebrates has been applied and validated. It is important to note thatimplementing the non-invasive, induction heating of nanoparticles in anRF field poses several technical challenges that have been addressed inthe studies performed to date and that these will also need to be takeninto account in in vivo studies. The RF field heats not onlymetallic/metal oxide nanoparticles but also the metal components of anystructure within the field. Therefore, the apparatus to be used forimaging, recording and behavioral monitoring must be designed so as toremove any metal components and replace them with ones that are inert inan RF field. Nonetheless, the design and optimization of the customequipment required to implement this technique is feasible.

In summary, a combination of nanoparticle heating in an RF field and atemperature responsive ion channel was used to convert localizedtemperature changes into remote, temporally controlled, and anatomicallydefined activation of specific cells. The NICE technology can furtherstudied in vitro and in vivo using two classical physiological processesrequiring specific calcium dependent functions: hormone release andneural activation. This technique can be applied in vivo to develop anovel method to regulate glucose metabolism and to examine thecontribution of specific neural populations to feeding.

Example 4 Analysis of NICE In Vitro

The use of NICE in vitro can be extended as follows: (i) examining thecellular responses to nanoparticles over time, (ii) validating theability of NICE to stimulate hormone release in vitro, (iii) modulatingneural activity in vitro, in hippocampal neurons or (iv) dopaminergicneurons in brain slices and (v) examining the combinatorial activationof distinct cell populations using a novel combination of epitope-taggedTRPV1 channel and functionalized nanoparticles tuned to heat at distinctfrequencies.

Cellular Responses to Nanoparticles and NICE.

Nanoparticles to be used in these studies will be prepared using welldescribed methods and optimized to reduce non-specific binding by use ofhydrophobic particles and functionalization with polyethylene glycol.Iron oxide nanoparticles will be synthesized using iron (III)acetylacetonate, reduced in the presence of oleylamine and oleic acid toyield monodispersed and single crystalline Fe₃O₄ nanoparticles with ahigh magnetic moment (Xu Zea 2009 Chem Mater 21:1778-1780 The resultinghydrophobic nanoparticles then are functionalized withcarboxyl-terminated polyethylene glycol (PEG-COOH), which will furthercovalent attachment of targeted biomolecules (streptavidin orantibodies) through EDC/NHS chemistry and reduce non-specific uptake bycells (Xie Jea 2007 Adv Mater 19:3163-3166) Gold nanoparticles will beprepared by a seed-mediated growth synthesis (Sau T, Murphy C 2004Langmuir 20:6414-6420, with the shaping surfactanthexadecyltrimethylammonium bromide replaced with thiolated PEG-COOHthrough an extensive aqueous-organic ligand exchange process. Wijaya A2008 Langmuir 24:9966-9969). Ligand exchange will be confirmed throughFourier transform IR analysis and zeta potential measurements.Nanoparticle characterization will take place through transmissionelectron microscopy (TEM), dynamic light scattering, X-ray diffractionand, in the case of gold, surface plasmon resonance to ensuremonodispersity and stability.

NICE utilizes cell surface tethering of nanoparticles for cellspecificity and to provide proximity for TRPV1 channel activation. Thefate of the nanoparticles after binding will be examined using bothquantitative and qualitative tools by inductively coupled plasma massspectrometer (ICP-MS) and microscopy, respectively. HEK 293t cellstransfected with TRPV1 and BAPTM will be incubated with thenanoparticles and, at 5 min, 30 min, 2 h, 4 h, 12 h and 24 h the cellswill be washed with Tween 20 to remove surface-attached nanoparticles.This supernatant as well as the homogenized cells will then be dissolvedin aqua regia and analyzed using ICP-MS. These experiments will give adetailed time-course of nanoparticle uptake over time. Localization andintegrity of the nanoparticles also will be investigated using TEM andconfocal microscopy. Transfected HEK 293t cells will be incubated withnanoparticles and at various times (as above), cells will be fixed andembedded. Cell sections (50-70 nm) will be stained with uranyl acetateand TEM analysis will be used to identify the location of nanoparticleswithin the section, using the high contrast of electron dense iron andgold, as well as further identification with electron-dispersivespectroscopy (EDS). Confocal micrographs also can be used to determinethe location of nanoparticles at the surface and within cells, usingfluorescently labeled streptavidin conjugated to iron oxide particles orthe dark-field light scattering of gold nanoparticles.

Initial studies indicate that NICE does not lead to apoptosis in NICEexcited or non-transfected cells. However, more detailed studies of cellviability following nanoparticle binding and RF exposure will beconducted on HEK 293t cells transfected with TRPV1 and BAPTM, incubatedwith nanoparticles before and following RF excitation. Mitochondrialfunction will be examined using the standard MTT assay Absorbance willbe read at 590 nm to quantify the mitochondrial reduction of yellow MTTto purple formazan. Membrane integrity following nanoparticle bindingand RF exposure will be assessed by colorimetric measurement of lacatatedehydrogenase activity released from damaged cells. The effects of theNICE protocol on DNA damage also will be examined using an in situapoptosis detection system Zoppi N et al. 2008 Biochim Biophys Acta1783:1177-1188). Quantitative PCR (qPCR) will also be performed on RNAextracted from transfected HEK 293t cells, with and without RF exposure,for markers of the Unfolded Protein Response (including C/EBP homologousprotein, phosphorylated inositol-requiring enzyme 1 alpha, eukaryoticinitiation factor 2 alpha and activating transcription factor3)(2-4)(2-4)(2-4)(2-4) need which are characteristic of ER stressKyriakakis et al. 2010 Cell Signal 22:1308-1316; Winnay et al. 2010 NatMed 16:438-445; Gjymishka A, et al. 2009 Biochem J 417:695-703

The effects of nanoparticle uptake by cells on the ability to activateTRPV1 and induce calcium entry also will be examined. Cells transfectedwith TRPV1 and BAPTM will be incubated with nanoparticles for increasinglengths of time, based on the studies above, such that initial timepoints demonstrate minimal NP uptake while later time points can includecells with intracellular nanoparticles. Cells will be loaded with thecalcium indicator, Fluo-4, and calcium entry in response to the RF fieldwill be measured. Further studies will examine the effect of NICE onneighboring cells by preparing two populations of transfected cells, oneexpressing BAPTM and binding Alexa 350-streptavidin iron oxidenanoparticles and one expressing TRPV1 and the fluorescent marker,mCherry. These cell populations will be mixed and loaded with Fluo-4before calcium imaging in the presence of the RF field. Absence ofcalcium entry in cells expressing TRPV1 and mCherry would indicate thatheating of nanoparticles bound to one cell does not affect adjacentcell.

Effect of NICE on Hormone Release from Cultured Cells In Vitro.

A classical calcium dependent process is hormone release from endocrinecells. The ability of NICE system to induce changes in hormone releasein vitro using stably transfected cell lines has tested. Constitutiveexpression was achieved using a CMV promoter to drive TRPV1 channelexpression and a beta actin promoter to drive BAPTM expression (FIG.25A). Insulin expression will be controlled by the Ca2+ sensitive NFATpromoter as described herein. Expression of TRPV1 and BAP will beconfirmed by immunocytochemistry (IHC). See FIG. 8.

The ability of NICE to modify endogenous insulin gene expression,synthesis and release will first be assessed using the RIN m5Finsulinoma cell line expressing TRPV1-BAPTM in vitro. On the day ofstudy, the stably transfected cells will be incubated in Kreb'sphosphate buffer (KRB) containing streptavidin coated 20 nm iron oxidenanoparticles for 30 min. Nanoparticle decorated cells will be exposedto the 465 kHz RF field and the effects will be assessed after 5 min, 10min, 30 min and 2 h, in part guided by the time course studies describedabove. Additional studies will examine the effect of repeated pulsed RFexposure for 5, 10 or 20 min periods for a total period of 2 h. Theseresults will guide the RF intervals used for combinatorial studies.Calcium imaging will be used to determine the time course of calciumentry and calcium induced insulin release into the media will bemeasured by ELISA with insulin gene expression examined by qPCR. Theeffects of RF field strength and duration on insulin secretion will beexamined. Control studies will examine the effect of TRPV1 and BAPTMexpression on insulin secretion in the absence of RF stimulation and theeffects of RF stimulation without addition of nanoparticles.

Calcium influx into cells can regulate gene expression via calciumdependent response elements. To assess the ability of NICE to modifygene expression, cells are engineered to express and release insulin ina Ca2+ dependant manner. As above, insulin is expressed downstream of apromoter with multiple Nuclear Factor of activated T-cell (NFAT)response elements and a minimal TATA promoter which has been shown toresult in a significant increase in expression in response to raisedintracellular calcium. This NFAT-TATA promoter is used to drive abicistronic construct with modified human insulin and luciferaseexpression (FIG. 1). Processing of proinsulin to insulin relies on twobeta-cell specific prohormone convertases, so a modified humanproinsulin with engineered furin cleavage sites is used as this isprocessed to mature insulin in non beta-cells both in vitro and in vivo(Shifrin A L, et al. 2001 Gene Ther 8:1480-1489) its release can bedifferentiated from endogenous murine insulin release in vivo. Thisconstruct is stably transfected into PC12 cells already expressingTRPV1His to generate PC12-NICE-Ins cells. Studies demonstrate thesecells secrete mature insulin. See FIG. 9.

The ability of NICE to induce insulin expression and release is studied.Cells are incubated with streptavidin iron oxide nanoparticles for thefinal 30 min of the preincubation period. The cells are then washed andplaced in a 465 kHz RF field and the effects on insulin expression andrelease are examined by qPCR and ELISA, respectively. The effects of RFfield strength and duration on gene expression and insulin release areexamined and optimized. Studies in HEK 293T cells transientlytransfected with TRPV1-BAPTM or TRPV1^(HIS) and NFAT insulin confirm theability of NICE to induce insulin gene expression and hormone release.See FIG. 8 and FIG. 6, respectively.

Effect of NICE on Electrically Excitable Cells In Vitro.

Calcium entry into electrically excitable cells induces depolarizationand initiates action potentials. NICE will be used to regulate theactivity of electrically excitable neurons in culture and in tissueslices in vitro. The ability of NICE to induce neural activity will beexamined in primary hippocampal cell cultures. These cells will beinfected with recombinant adenovirus expressing TRPV1-BAPTM constructsand then incubated with streptavidin-coated nanoparticles. Nanoparticledecorated cells will be exposed to the RF field and the effects assessedby calcium imaging and whole cell patch clamping. Preliminary studiesindicate electrophysiological recording in the electromagnetic field isfeasible. These studies will provide details of the intracellularcalcium concentration achieved, the temporal resolution of the calciumentry in neurons, the degree of depolarization and spike activityachieved with NICE, and the effects of multiple RF exposures. Currentsinduced by RF activation will be measured while holding the neurons involtage clamp. Membrane depolarization and spikes will be determined incurrent clamped neurons to determine the kinetics of activation andprecision of control. Additional control experiments will examine theeffect of TRPV1 and BAPTM expression on electrical properties of neuronsin the absence of RF stimulation, the effects of RF stimulation withoutnanoparticle binding and to determine if there is a bystander effect incells adjacent to those decorated with nanoparticles. In addition, thestrength of the RF will be titrated to find the minimum field strengthrequired for inducing an action potential.

Assays of the Effects of NICE in Brain Slices Ex Vivo.

The ideal neuronal activation strategy provides anatomical specificitythat, for the NICE system, would require expression of TRPV1 and BAPTMin a defined neural population. A recombinant adenovirus expressing aFlip-excision construct (Atasoy D, et al. 2008 J Neurosci 28:7025-7030)will be used to achieve cre-dependent expression of TRPV1 and BAPTM(Ad-FLEX-NICE) (FIG. 25C). In this construct, in the absence of crerecombinase, TRPV1 and the beta actin promoter will be in an invertedorientation and inactive. In the presence of cre, TRPV1 and the betaactin promoter return to the correct orientation and are locked in placeby the excision of the additional lox site. This system will allow cellspecific expression of the TRPV1 and BAPTM in cells that express crewith little or no expression in cells that do not express cre.Transgenic mice expressing cre recombinase in dopamine transporterneurons (DAT-cre) will be crossed to a reporter strain,ROSA26-lox-stop-lox-EYFP mice (Ekstrand M I, et al. 2007 Proc Natl AcadSci USA 104:1325-1330) DAT-cre/YFP mice (age 4 weeks) will receive astereotactic injection of high-titer Ad-FLEX-NICE stocks (˜150 nl ofstock of 108 infectious units) into the ventral tegmental area (VTA). Ina subset of animals, expression of the construct will be determined bytriple IHC to confirm co-localization of YFP (expressed solely in creneurons), TRPV1 and Alexa-streptavidin binding. Wild-type mice also willbe injected to ensure there is no expression of the cre dependentconstruct in the absence of cre recombinase. Ten to 14 days after viraldelivery, mice will be sacrificed and sections (200 μm) will be cut.After resting, slices will be incubated with streptavidin-coated ironoxide nanoparticles for 30 min. The electrophysiological properties ofinfected, decorated neurons will be determined by patch clamp recordingin the absence and presence of RF stimulation. Overall, these studieswill confirm the ability of NICE to stimulate Ca2+ influx and neuralactivity in neural slices in vitro.

Combinatorial Activation of Specific Cellular Populations. The NICEsystem will be refined further to allow combinatorial activation ofspecific multiple cell populations, even if they are interspersed, usingepitope tagged TRPV1 channels. Nanoparticles of different compositionsand shapes heat at different EM power and frequency with differentrates. For example, iron oxide nanoparticles are heated by a frequencyof 465 kHz which does not heat gold, whilst gold nanoparticles heatrapidly at a frequency of 13.5 MHz. Furthermore the responsecharacteristics of the particles also are influenced by their size.Thus, gold could be activated at a much lower RF field strength thaniron oxide (i.e., gold heating faster than iron oxide) (Saleh S. et al.J. Biomedical Science and Engineering, 2008, 1, 182-189) Hence, even inthe presence of both gold and iron oxide nanoparticles it is possible toselectively activate gold. Uniquely modifying TRPV1 channels expressedunder the control of cell-specific promoters, provide distinct cellsurface tags to direct nanoparticle binding. Particles can bespecifically targeted to different cell types by coating them withrecombinant antibodies directed against short epitopes. This can allowdifferential regulation of distinct cell populations in the sameanatomical region. Specific antibodies recognizing a library of shortlinear epitopes that have been identified using phage display will beused. These epitopes will be inserted in frame into the firstextracellular loop of TRPV1 which has been shown can be modified withoutaltering the response characteristics of this channel.

Two different nanoparticles will be targeted: iron oxide (465 kHz) andgold nanoparticles (13.5 MHz) (Moran C, et al. 2009 Nano Research2:400-405) to two different cell populations (FIG. 26).Carboxyl-terminated nanoparticles will be functionalized withrecombinant single chain Fv antibody fragments (scFv) (Vigor K L et al.2009 Biomaterials) to the specific short linear epitopes, His6× (6aa)and FLAG (8aa). Constructs will be generated in which these epitopes areintroduced into the 1st extracellular loop of the TRPV1 channel. Asmentioned above, it has been shown that this does not interfere with thetemperature sensing properties of the channel. Each construct will betransfected into HEK 293t cells and paired with a distinct NFAT-REdriven luciferase reporter (CBRluc—red luciferase, CBG99luc—greenluciferase) to give two different reporter read-outs of calcium entryfor each cell type. Post-transfection, the two cell lines will be mixedand cultured together for a further 48 h before incubating in serum freemedium overnight. On the day of study, the cells will be incubated witha cocktail of two nanoparticles; iron oxide functionalized withanti-His6× and gold nanoparticles functionalized with anti-FLAG. Thelocalization of different particles to different cells will be confirmedusing IHC. The cells will then be exposed to sequential pulses (5 min,10 min or 20 min) of RF frequencies of 465 kHz and 13.5 MHz using avariable frequency amplifier for a total of 40 minutes at each frequencybefore the cells are lysed and individual luciferase activity measured.

These studies will make use of existing nanoparticle technology butnanoparticles of differing chemistries (e.g., iron oxide and gold), size(5-50 nm), and shape (spheres, rods) also will be developed to produce arange of distinct RF excitation profiles and power-heating profiles toimprove this combinatorial approach. These in vitro studies provideevidence that the technology is able to activate ensembles of two ormore different classes of cells or neurons alone or in combination evenif these cells are colocalized in a small anatomical region.

Example 4 In Vivo Studies of NICE on Hormone Release

The goal of an artificial endocrine organ has yet to be achieved but theability to remotely control hormone expression and release can provide asignificant advance towards this aim. Once the effects of NICE oninsulin synthesis and release have been optimized in vitro, in vivostudies to examine the effects of NICE on insulin release will becarried out.

The power required to create an RF field increases with the square ofthe coil diameter and thus a significantly more powerful generator(50-75 kW) will be required for these in vivo studies as compared tothose required in vitro. Generators with sufficient power are readilycommercially available (Ultraflex Power Technologies, NY). The size ofthe behavioral chamber will be kept to a minimum to reduce the powerrequired whilst ensuring the mouse is able to move freely. Studies willfocus on the design of both the coil and the plexiglass chamber used forassessment to ensure a uniform RF field is achieved. Modifications ofthe coil and generator will be made pending the results of thesestudies.

Effect of NICE on Insulin Expression in Implanted Cells In Vivo.

Initial studies will examine the ability of NICE to regulate calciumdependent modified insulin expression in vivo as an indication ofwhether NICE can be employed to regulate gene expression and hormonerelease. Stably transfected PC12-NICE-Ins cells expressing TRPV1, BAPTM,and NFAT driven modified insulin will be used since these cells cansynthesize and secrete insulin and are capable of forming subcutaneoustumors in immunodeficient mice (Fritz M D et al. 2006 Exp Cell Res312:3287-3297). These stably transfected cells will be injectedsubcutaneously into nude mice to create tumors expressing TRPV1, BAPTM,and NFAT-insulin IRES luciferase constructs. This will be confirmed byIHC. Freely diffusable iron oxide nanoparticles (20 nm) or vehicle willbe injected into the tumor and the effects of RF stimulation on bloodglucose and insulin, and glucose tolerance will be examined. Insulinexpression will be determined by qPCR of tumor RNA in a subset of mice.Luciferase expression, as an indicator of calcium entry, in tumors willbe confirmed by luminometer studies in vivo (Birsoy K. et al., 2008 ProcNatl Acad Sci USA 105:12985-12990). Further studies will focus on theeffect of stimulating PC12-NICE-Ins cells on glycemic control andglucose tolerance in streptozocin-treated diabetic nude mice. Initialstudies, based on in vitro studies described above, will optimize thetime period of RF exposure, by examining these measures after 5, 10, 20or 40 mins of RF exposure. Insulin and glucose levels will be measuredimmediately following RF stimulation and at 10 min, 30 min, 1 h and 2 hthereafter to follow the time course of the insulin response tostimulation. In this, and all in vivo studies, a number of mice will beexamined in detail post-mortem for pathological changes that may beassociated with nanoparticle administration or RF exposure.

Effect of NICE on Endogenous Insulin Release In Vivo.

For studies examining the effect of NICE on endogenous insulin release,transgenic mice will be used to express the NICE components. Twotransgenic mouse lines will be generated to overcome the lowerexpression of the second gene after an IRES. The first will use aninsulin promoter to drive TRPV1 expression (FIG. 27A) and the secondwill use the insulin promoter upstream of BAPTM (FIG. 27B). These micewill be crossed resulting in TRPV1 and BAPTM expression exclusively inbeta cells (FIG. 27C). In addition, a further cross to a reporter mouseexpressing luciferase downstream of NFAT response elements and a minimalpromoter results in co-expression of calcium dependent luciferase (FIGS.27D and E). Immunohistochemistry will be used to confirm TRPV1 and BAPTMexpression in beta cells.

These studies aim to address whether NICE can be employed to regulateendogenous insulin release in vivo without the necessity of injectingcultured cells. At 8-10 weeks, transgenic mice will receive pancreaticinjections of iron oxide nanoparticles or vehicle. Nanoparticle bindingto beta cells will be confirmed using double IHC for insulin andstreptavidin. It has been established that the 20 nm particles that willbe used can freely diffuse in tissues. Once the optimal time forparticle binding to the cells has been determined, the effect of RFfield stimulation on blood glucose and insulin will be examined. Aluminometer will be used to examine the luciferase response to RFstimulation in vivo as previously shown by us. The effects of RF fieldpower and duration on these parameters will be studied at various times(see above). When these parameters have been optimized, mice also willundergo glucose tolerance testing in the presence or absence of RFstimulation. Pending these results, these studies will be repeated oncontrol and InsTRPV1-BAPTM mice treated with streptozocin (a model ofinsulin deficient diabetes), high fat diet (a model of insulin resistantdiabetes) or transgenic mice crossed to leptin deficient, glucoseintolerant, ob/ob mice.

Combinatorial Activation of Nanoparticles to Regulate Glucose MetabolismIn Vivo.

Combinatorial activation of nanoparticles can be applied to develop asystem whereby circulating glucose can be modulated through the releaseof either insulin or glucagon. PC12 are stably transfected to give twopopulations, the first expressing TRPV1 modified with a His6× tag andNFAT driven insulin expression (PC12-TRPV1His-Ins) and the secondexpressing TRPV1 modified with a FLAG tag and NFAT driven glucagonexpression (PC12-TRPV1FLAG-Glucagon). A combination of the two celllines are then injected subcutaneously into nude mice to give a tumorwith a mixed cell population of both PC12-TRPV1His-Ins andPC12-TRPV1FLAG-Glucagon which can be confirmed by IHC for His and Flagepitopes. The tumor is then injected with a cocktail of iron oxidenanoparticles functionalized with anti-His6× antibody and goldnanoparticles functionalized with anti-FLAG antibody. RF stimulation at465 kHz should heat iron oxide nanoparticles and stimulatePC12-TRPV1His-Ins cells to synthesize and release insulin reducingplasma glucose since gold nanoparticles are not heated at this lowerfrequency. RF stimulation at 13.5 MHz preferentially heats goldnanoparticles targeted to activate PC12-TRPV1flag-Glucagon cells torelease glucagon and increase plasma glucose. Finally, alternating 10minute pulses of RF frequencies of 465 kHz and 13.5 MHz using a variablefrequency amplifier will be used to stimulate the release of bothglucagon and insulin to modulated plasma glucose. These studies allowone to confirm that NICE can be used to activate different cell typesalone or in combination. These studies allow one to remotely up regulateor down regulate blood glucose upon which future development ofartificial endocrine organs may build on. See FIG. 10 for demonstrationof in vivo regulation of insulin expression.

Example 5 Effects of NICE on Neural Activity In Vivo

Following optimization of NICE on neural activity in vitro, its efficacyin mice in vivo will be confirmed. To this end, the utility of NICE willbe demonstrated by studying a well-characterized neural network andbehavior; the dopamine reward pathway using a self-stimulation paradigmand compare its actions to those of an invasive neural stimulationprotocol using channelrhodopsin and blue light. The use ofchannelrhodopsin has been validated to induce reward behavior, seebelow, and this system will be used to compare the ability of the NICEprotocol to induce this same behavior.

Self Stimulation Protocol Using Channelrhodopsin.

In traditional self-stimulation protocols, rodents are trained toassociate lever pressing with delivery of an electrical stimulus into aspecific brain region. The behavioral chamber consists of a choicebetween two levers, one lever results in direct brain stimulation whilethe other has no effect. A decreased latency to lever press andincreased frequency of lever presses indicates stimulation of the brainregion is rewarding to the animal. Using such a protocol, it has beendemonstrated that stimulation of the ventral tegmental area (VTA), richin dopamine neurons, is rewarding (Fibiger H C, 1987 J Neurosci7:3888-3896; Druhan J P, Fibiger H C, Phillips A G 1990 Behav Brain Res38:175-184)(8, 9)(8, 9)

Recently, a modified version of this self stimulation protocol has beenvalidated using a lickometer to trigger light dependent opening ofchannelrhodopsin and thus specific activation of dopaminergic neurons.The lickometer places a sipper tube behind a “>” shaped plate such thatthe mouse is forced to lick for water delivery. An infrared beam placedbetween the plate and the sipper counts each beam break by the tongue asthe animal licks for water. In these studies, the operant behavior isconsumption of water from the lickometer under conditions where the micehave a choice between one lickometer port which has no effect, and asecond port which results in light delivery activating VTA neurons thatexpress ChR in dopamine transporter (DAT) cre cells. This system confersanatomical specificity by expressing the effector, channelrhodopsin(ChR2), only in a defined neural population i.e.; the dopaminergicneurons expressing a DAT-cre construct expressed in these neurons. Thescheme for this experiment is shown in FIG. 28. In studies using thissystem, mice with dopamine neuron specific expression of ChR2 show asignificant increase in consumption from the light-activating lickometerport (FIG. 28). This paradigm provides a means for comparing therobustness of light vs. RF for neural activation of this rewardbehavior.

Self Stimulation Protocol Using NICE.

The effects of NICE on dopamine neuron excitation will be directlycompared with those already achieved by optogenetic stimulation mediatedby channelrhodopsin (ChR2). In this manner, a baseline stimulation isprovided that is achieved currently using invasive light activation andallows one to assess the efficacy of RF stimulation. Stereotacticinjection of Ad-FLEX-NICE or AAV-FLEX-ChR2 encoding cre-dependentconstructs into the VTA of DAT-cre mice will be used to drive specificexpression of NICE constructs (TRPV1 and BAPTM), ChR2-mCherry or mCherryalone (control) in the dopamine neurons of DAT cre mice.

Ten days after virus injection, ChR2 injected and control mice will beimplanted with a fiber optic system to deliver blue light (473 nmwavelength) to the VTA and allowed to recover prior to training NICEmice and a second group of control mice will receive a VTA injection ofstreptavidin-coated iron oxide nanoparticles. The location of thenanoparticle injection will be confirmed by MRI, which can be used toprecisely localize the beads. The distribution of particles ondopaminergic neurons will also be analyzed using double IHC forstreptavidin and dopamine.

Following acclimation to the behavioral equipment (Med Associates), micewill be given three daily 60 min training sessions to establish theassociation between one of two lickometers (L-A or L-B; counterbalanced)and intracranial stimulation either via a pulse of blue light (ChR2) ora pulse of RF field (NICE). For half the animals, L-A will be rewardedand L-B unrewarded and for half, L-B will be rewarded and L-Aunrewarded. Consumption from the lickometer at each training session andat testing will be recorded by the infrared monitoring system. Havingacquired the association between the lickometer and delivery of light orRF field, retrieval will be tested 2 days after training The latency toconsumption and the number and pattern of licks will be recorded over a2 h trial period. A decreased latency and increased frequency to lick atthe port associated with RF delivery would suggest that NICE is capableof activating DAT cre neurons to modify reward behavior. At the end ofthe study, mice will be sacrificed and specific expression ofTRPV1-BAPTM (anti-TRPV1, anti-tyrosine hydroxylase (TH) andAlexa-streptavidin) or ChR2 (mcherry, anti-TH) in the DAT neuronsconfirmed by immunohistochemistry.

As with in vitro studies, the RF field will heat metal and therefore thebehavioral equipment must be free of all metal components. This will beachieved by using a custom-built plexiglass chamber with glass sippersand fiber optical cabling delivering information from the infrared beambreak system. A video system mounted above the RF field will monitormovement in the chamber. The chamber will be encircled by a custom-madecoil sufficient to provide a uniform RF field throughout the chamber andto a height equivalent to that of a rearing mouse.

Overall, this set of studies will confirm that RF field stimulation iseffective at neural stimulation to modify behavior in vivo invertebrates. It also will allow comparisons of the ability of NICE toactivate dopamine neurons for self-stimulation in comparison to lightstimulation via ChR2. Additional studies may be performed to optimizethe field strength and duration of the individual stimulus deliveredwith each response and thereby create response-field strength curves andresponse-duration curves. These studies will allow optimization of theNICE system in vivo in a well-defined paradigm alongside existingstimulation tools.

Combinatorial Activation of Nanoparticles to Study Feeding Behavior InVivo.

The use of NICE is most advantageous for the study of complex behaviorswhere intracranial implants and tethering to deliver light or electricalstimuli may subtly alter or hinder behavior. One such behavior isfeeding, which is particularly susceptible to interference such thateven relatively low levels of stress may alter feeding patterns (AbbottC R et al. 2006 Int J Obes (Lond) 30:288-292). The hypothalamusregulates feeding to maintain body weight in a narrow range whilehedonistic systems, particularly dopamine reward pathways and corticalregions, modulate and may even over-ride this (Davidson T L 1993 PsycholRev 100:640-657). The hypothalamic arcuate nucleus is known to be a keycomponent of hypothalamic feeding circuitry. The arcuate contains twoprimary cell populations expressing the leptin receptor, oneco-expressing neuropeptide Y (NPY) and agouti related peptide (Agrp) andthe second co-expressing the gene pro-opiomelanocortin (POMC) whosemajor product is alpha melatonin stimulating hormone (α-MSH) (Cone R D,2001 Int J Obes Relat Metab Disord 25 Suppl 5:S63-S67) Central andarcuate Agrp injection potently stimulates feeding, knock-down in Agrpexpression reduces body weight and post-natal loss of Agrp neuronsreduces food intake and body weight (Bewick G A et al. 2005 FASEB J19:1680-168; Gropp et al. 2005 Nat Neurosci 8:1289-1291; Rossi M. et al.1998 Endocrinology 139:4428-4431; Makimura H. et al. 2002 BMC Neurosci3:18). Conversely, central administration of α-MSH inhibits feeding andmice lacking the MC4 receptor, at which α-MSH is an agonist arehyperphagic and obese (Shimizu H, et al. 1989 Life Sci 45:543-552;Huszar D. et al. 1997 Cell 88:131-141). However, these neurons alsoexpress classical neurotransmitters and other neuropeptides whoseeffects may not be reflected in the pharmacological approaches ofintracranial peptide delivery or peptide over-expression. Further, therelative dominance of these neural populations on feeding is unknown forexample, whether stimulation of Agrp neurons outweighs the effect ofstimulating POMC neurons or vice versa. Until now, the technology hasnot been available to acutely stimulate defined cell populations andexamine the consequences in freely moving animals. Therefore, NICE willbe used to selectively stimulate these important neuronal populations invivo, either individually or together, to examine their physiologicalroles in feeding.

BAC transgenesis will be used to generate mice expressing epitope-taggedTRPV1 channels directly under the control of the Agrp promoter (Histagged) or the POMC promoter (FLAG tagged) (FIG. 11) (Gong S et al. 2002Genome Res 12:1992-1998). When these mice are crossed, dual transgenicprogeny expressing Agrp-Trpv1His and POMC-Trpv1FLAG will be generated(FIG. 29C). Specific expression will be confirmed by dual IHC forHis/Agrp and FLAG/α-MSH. Mice will receive a stereotactic injection intothe arcuate nucleus of a mixture of anti-His coated iron oxidenanoparticles and anti-Flag gold nanoparticles. These will bindspecifically to Agrp neurons and POMC neurons respectively. The effectsof neural stimulation on food intake will be determined in a custombuilt, metal-free metabolic chamber (Columbus Systems). A coilsurrounding the chamber will generate a variable RF field and thepattern, frequency and total food intake will be examined along withfeeding related behaviors such as activity.

First, feeding behavior in response to stimulation of individual neuralpopulations will be examined. The effects of Agrp neuron stimulationwill be examined in the presence of RF field stimulation at 465 kHz toheat iron oxide particles (FIG. 29E). The effects of POMC neuronstimulation will be with RF stimulation at 13.5 MHz which selectivelyheats gold nanoparticles (FIG. 29F). Finally, combinatorial stimulationwill be achieved by pulsed alternating RF stimulation at 465 kHz and13.5 MHz (FIG. 29G). These studies will provide important data regardingthe physiological roles of these two neural populations. Overall, thesestudies will employ NICE to dissect the contribution and the interactionof specific neuronal populations to a complex behavior, feeding, infreely moving animals. These studies also provide a basis for using themethod to probe the role of other neural populations to control feedingand other behaviors.

In summary, a combination of nanoparticle heating in an RF field withdefined expression of a cell surface tag for targeted nanoparticlebinding will be used together with a temperature responsive ion channelto convert localized temperature changes into remote, temporallycontrolled and anatomically defined cell activation. Using the currentin vitro system validation of this technique has been conducted to showthat NICE results in a significant increase in intracellular calcium andcan thereby stimulate calcium dependent gene expression. The cellularresponses to NICE will be examined and validation combinatorial cellactivation. The novel technology will be used to modulate hormonerelease in vitro and in vivo and to modify neural activity in vitro andin vivo to investigate feeding behavior. Together these studies willfurther support the use of NICE as a tool for external, non-invasive,specific cell excitation and the employment of NICE as a uniquemethodology to investigate the effects of specific cell stimulation.

Example 6 Cell Based Nanoparticle Therapy for Diabetes

Transfecting cells with the ferritin construct resulted in thedistribution of nanoparticles throughout the cell cytoplasm and with anaverage distance of the ferritin nanoparticle to the TRPV ion channelapproximately equal to the cell radius. Previous mathematical modelingpredicts that particles within 200 nm would lead to sufficient heatingto trigger channel activation (Huang et al., Nat. Nanotechnology5:602-606). The number of ferritin nanoparticles were thereforequantified within 200 nm of the cell membrane by EM. It was found thatthere are 12.6±2.86 particles per 0.2 μm² with an average distance tothe cell membrane of 60.3±2.85 mn. This distance is not dissimilar fromthe average distance between nanoparticles bound to transmembrane biotinacceptor protein tether (100 nm). Assuming the ferritin particles areevenly distributed through the cell, this would give 5×10⁸ ferritinparticles/cm³. This is in considerable excess of the 1.1 pg of iron(5×10⁵ nanoparticles assuming ferritin has a similar composition to ironoxide nanoparticles but 10 nm diameter) calculated to be necessary toachieve a 5° C. increase in temperature.

Results

Diseases related to tissue loss have traditionally been treated bypharmacological replacement of cell secretory products, such as insulinor dopamine, and more recently by organ or cell transplantation fromcadaveric donors. Pharmacological therapies often have side effects andeffective means of delivering peptide drugs in particular are limited.However, more physiological treatments such as allogenic cellimplantation are hampered by lack of donors and the necessity of longterm immunosuppression. Therefore, a technique which allows precise,remote activation of defined cells to trigger gene expression andpeptide release in vivo offers potential as a cell replacement therapyas well as a tool for basic research.

The importance of developing means for activating specific cells hasbeen widely appreciated as evidenced by the use of direct stimulationthrough implanted electrodes. However this approach is limited byvariable activation, the need for permanent implants, and tissue damage.In addition this technique does not allow selective activation ofspecific cells. An alternative approach makes use of drug induciblesystems to alter gene expression or ion channels to modulate cellactivity. While drug inducible gene expression systems do allow cellularspecificity, they typically have a relatively slow onset of action. Incontrast, ion channels rapidly regulate intracellular ion concentrationsby allowing the selective passage of cations or anions, and in turnmodulate intracellular functions in a cell specific manner. The use ofion channels has many advantages; their structure and function arerelatively well described, they have a rapid time course of activationand a broad range of channels exist in mammalian and non-mammaliancells, which may be exploited in the search for the optimum means ofmodifying cellular activity. Recently, the non-mammalianchannelrhodopsin (ChR2) gene, a light activated cation, has beenemployed to rapidly activate molecularly defined neurons when exposed toblue light. This system gives anatomical specificity and temporalcontrol but requires fiber optic light delivery via invasive chronicimplanted devices because light penetration is relatively poor. Thus ameans for activating ion channels without the requirement of animplanted device could represent an important potential means foractivating cells. Describe herein is a means for non-invasive excitationof a defined cell population in vivo using a radiofrequency field toheat iron oxide nanoparticles which in turn activate the temperaturesensitive TRPV1 channel to trigger calcium entry. Calcium influx in turnactivates gene expression and in the current system, hormone release.This approach has been validated by modulating the expression andsecretion of the peptide hormone, insulin, and shown that it can be usedto lower blood glucose in mice.

Remote Activation of Cells Using Metal Nanoparticles and an EpitopeTagged Temperature Sensitive Channel.

In order to activate cells and tissues non-invasively, it is necessaryto have a signal that is capable of passing through tissue.Radiofrequency (RF) signals at low and medium frequencies penetratetissues freely and without significant energy absorption. In contrast,metallic/metal oxide nanoparticles placed in an alternating RF fieldabsorb energy and heat in a controlled manner dependent on particle size(FIG. 12) and the field strength. In vitro, the temperature responseachieved is fast and decays quickly (inverse of the square of thedistance) thus potentially providing a rapid, highly localized ‘on-off’switch. Metal nanoparticles can readily be functionalized to target todefined cell populations by coating with specific antibodies thatrecognize proteins that are normally expressed on a cell or transfectedinto that cell. Thus, nanoparticles are well suited for inducing cellsurface temperature changes that can be transduced into cellularresponses by temperature sensitive channels in vitro and in vivo.

The local temperature change achieved by exposing nanoparticle-coatedcells to radiowaves can be transduced into calcium entry by targetedexpression of TRPV1 channel. This single component, cell surface cationchannel detects small changes in temperature just above thephysiological range (>42° C.) and undergoes a conformational changeallowing calcium entry. The response is proportional to the temperaturechange and relatively rapid, thus transducing temperature variationsinduced by the heated nanoparticles into a graded calcium current withattendant cell activation. In addition these channels do not desensitizeafter repeated activation as repeated heating potentiates calcium entry.

The method described herein makes use of metal nanoparticles, directedto specific cells, and TRPV1 ion channels to remotely stimulate cellactivity and gene expression as follows. A single construct encodingTRPV1, modified to incorporate a unique extracellular His×6 epitope tagwas expressed in specific cells (TRPV1His). Iron oxide (IO)nanoparticles functionalized with monoclonal antibodies against theHis×6 epitope tag are targeted to these cells. In the presence of a RFfield, local heating of IO nanoparticles above the threshold for TRPV1channel activation (42° C.) triggers calcium entry and cell activation(FIG. 1). The increase in intracellular calcium then activates geneexpression under the control of a calcium dependent NFAT promoter inwhich the calcium activated protein serine/threonine phosphatase,calcineurin, dephosphorylates the regulatory domain of NFAT, (nuclearfactor of activated T cells), allowing its translocation into thenucleus where it activates the transcription of genes downstream of NFATresponse elements.

Reports have described a two component system using a biotinylatedtransmembrane protein as a nanoparticle anchor and a separate TRPV1 toinduce calcium entry, hereafter called V1 BAP (FIG. 8A). A singlecomponent system was benchmarked against this dual component system andboth systems were used to control gene expression and hormone release invitro before using the novel single component system to regulate geneexpression and hormone release in vivo.

Nanoparticle Decoration of Cells In Vitro.

A modified TRPV1 channel was used as both a nanoparticle tether andeffector. A short epitope tag, His×6, was introduced into the firstextracellular loop of TRPV1. Modification did not significantly alterthe temperature sensing ability of TRPV1 (FIG. 31). This site is exposedto extracellular signals but distant from putative temperature andvoltage sensing sequences and the pore region of TRPV1. Modifying thechannel to act as the tether offers several advantages. Firstly, byincreasing the proximity of nanoparticle binding and temperature sensingthe effect of heat dissipation from the nanoparticle is reduced. Thismay reduce the density of nanoparticle binding required to achieve TRPV1opening. Secondly, a single construct is readily targeted to definedcell populations using defined promoters. Finally, conditionalexpression can be controlled by recombinases such as cre or Flp.

Functionalized iron oxide nanoparticles were used to decorate the cells.Size dependent heating of IO nanoparticles occurred at 465 kHz withmaximum heating achieved using an aqueous solution of 20 nm diameterparticles. With an alternating magnetic field (465 kHz,), an increase ofup to 17° C. can be achieved in a suspension of 20 nm iron oxideparticles (1 mg/ml) (FIG. 2A) with an initial heating rate of 0.15°C./s. A 5° C. rise in nanoparticle temperature above body temperature issufficient to open TRPV1 channels and can be reached in 30 s.Nanoparticles of this size are within the limits of the extracellularspace and therefore able to diffuse. IO nanoparticles (20 nm)functionalized with monoclonal anti-His antibody bind to TRPV1Hisexpressed in transfected human embryonic kidney (HEK 293) cells. Thereis a significant increase in nanoparticle binding in cells transfectedwith TRPV1His (19.78±2.23 nanoparticles per mm cell membrane) comparedto untransfected cells (2.85±0.32 nanoparticles per mm cell membrane)(FIGS. 30A and B) and greater than the density achieved withstreptavidin functionalized IO nanoparticles bound to cells expressingthe two component system, TRPV1 BAP (9.07±2.85 nanoparticles per mm cellmembrane). In these studies, the results of the one component system (anepitope tagged TRPV1) was compared to a previously published twocomponent system that uses a biotinylated transmembrane protein as ananoparticle anchor and a separate TRPV1 to induce calcium entry,hereafter called V1 BAP (FIG. 8A) were compared. This two componentsystem has been tested in vitro.

Nanoparticle Activated Insulin Expression and Release In Vitro.

In order to assess the potential anti-diabetic effects of this system,the modified TRPV1 construct and an NFAT-insulin construct wasintroduced into PC12 cells as well as 293T cells. These cells processand secrete proteins in response to increased intracellular Ca++. Asynthetic promoter comprised of three calcium response elements: serumresponse element (SRE), cyclic AMP response element (CRE) and nuclearfactor of activated T-cell response element (NFAT RE) and a minimalpromoter were cloned upstream of a modified, furin sensitive insulincDNA (FIG. 19A). Processing of proinsulin to insulin relies on two betacell specific prohormone convertases so a modified human proinsulin withengineered furin cleavage sites was used. This expressed protein isprocessed to mature insulin in non beta cells both in vitro and in vivo(Shifrin et al., gene Ther. 8:1480-1489) and its release can bedifferentiated from endogenous murine insulin release in vivo. Like betacells, cells expressing furin sensitive insulin synthesize bothproinsulin and insulin and the ratio of proinsulin to insulin variesboth with cell type and basal vs activated state.

HEK 293t cells expressing calcium dependent human insulin and eitherTRPV1His or TRPV1 BAP were decorated with functionalized JOnanoparticles. Applying a RF magnetic field to nanoparticle-decoratedcells expressing TRPV1His or TRPV1 BAP and calcium regulated furinsensitive insulin significantly increased proinsulin release (FIG. 19B)(TRPV1His: control 100±13.94% basal, RF treated 671±234.9% basal. TRPV1BAP: control 100±23.96% basal, RF treated 477.4±136.1% basal) andinsulin gene expression (FIG. 19C) (TRPV1His: control 1.0±0.18, RFtreated 2.20±0.53 relative gene expression, p<0.05. TRPV1 BAP: control1.0±0.06, RF treated 2.06±0.38 relative gene expression, p<0.05). Theincreases in proinsulin release are blocked by the non-specific TRPchannel inhibitor, ruthenium red (FIG. 19B). There was a trend towardsan increase in proinsulin release after 15 mins of RF treatment,presumably initially through the release of preformed insulin containingvesicles and with a significant increase in release at 1 hour (FIG.19D), whilst insulin gene expression begins to increase after 45 minutesand also becomes significant at 1 hour (FIG. 19E). This time course issimilar to the expression of c-fos, a gene whose expression is alsocalcium dependent.

The effects of RF dependent heating of IO nanoparticles were confined todecorated cells since there was no release of proinsulin when cellsexpressing BAPTM as a nanoparticle tether are mixed with, and thereforeadjacent to, cells expressing TRPV1 and calcium regulated furinsensitive insulin (FIG. 19F) (Proinsulin release: control 100±78% basal,RF treatment 109±38.2% basal). Proinsulin release only occurs when allcomponents of the system are present: TRPV1, nanoparticles and RFmagnetic field. No significant increase in proinsulin release wasobserved in HEK 293t cells decorated with IO nanoparticles in thepresence of an RF magnetic field without TRPV1 (transfected with BAPonly), in cells with TRPV1 but without nanoparticle binding (transfectedwith TRPV1 only), or in cells with nanoparticles and TRPV1 (TRPV1His orTRPV1 BAP) but in the absence of the RF magnetic field (FIG. 32).

To assess cell viability, immunohistochemistry was used to quantify twomarkers of apoptosis-active caspase 3 and terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL). No differences inimmunopositive cell counts were observed between TRPV1His transfected239t cells treated with nanoparticles alone and those treated withnanoparticles and RF magnetic field application (FIGS. 19G and H)(TUNEL: control 123±37 cells, RF treatment 124±17 cells. ActivatedCaspase-3: control 136±21 cells, RF treatment 163±11 cells).

Nanoparticle Regulation of Blood Glucose In Vivo.

Experiments were conducted to translate in vitro findings in vivo bytesting whether the remote activation of proinsulin gene expression,insulin synthesis and release could modulate blood glucose in mice.Stably transfected PC12 cells expressing TRPV1His and calcium regulatedfurin sensitive insulin were established. As above, this endocrine cellline is capable of synthesizing and secreting mature insulin. In vitrostudies examining the effects of RF treatment on proinsulin release andinsulin gene expression replicated the findings in transfected HEK 239tcells (FIG. 33A-C). Stably transfected PC12-TRPV1His-Ins cells wereinjected subcutaneously into the flank of nude mice and formed tumorsexpressing TRPV1His (FIG. 33D) and furin sensitive insulin constructs.Following an overnight fast, PBS or IO nanoparticles were injected intothe tumors of anesthetized mice (50 ul total volume, nanoparticleconcentration 8 mg/ml). Blood glucose and plasma insulin were measuredbefore, during and after the application of an RF field (See FIG. 33E).Applying the RF magnetic field resulted in significant decrease in bloodglucose in mice treated with IO nanoparticles compared with thosereceiving PBS followed by RF treatment (FIG. 10A)(Δ Blood glucose at 30mins: PBS control −10.99±9.72, nanoparticle treated −53.62±8.90, p<0.05.At 45 mins: PBS control −9.74±8.52, nanoparticle treated −60.88±11.59,p<0.05. At 60 mins: PBS control −6.24±15.29, nanoparticle treated−55.12±13.15, p<0.05). There was a significant difference in thecumulative change in blood glucose between PBS and nanoparticle treatedmice over the course of the study (FIG. 10B) (TRPV1His, NF Ins AUC(0-120 min): PBS treated 271.7±691.6, nanoparticle treated −2695±858.3mgdl-1 min, p<0.05). Plasma insulin was also significantly increased inmice receiving nanoparticle injection and RF magnetic field treatmentbut remained unchanged in control mice injected with PBS (FIG. 10C)(Plasma insulin: PBS (−30 min) 1.83±0.380 μIU/ml, PBS (30 min)1.75±0.360 μIU/ml, nanoparticles (−30 min) 2.26±0.760 μIU/ml,nanoparticles (30 min) 3.25±0.640 μIU/ml, p<0.05). In addition,proinsulin mRNA levels were significantly higher in nanoparticleinjected tumors treated with RF magnetic field compared to thoseinjected with nanoparticles alone (FIG. 10D) (Control: 1.0±0.2 relativeinsulin gene expression, RF treated: 2.03±0.3 relative insulin geneexpression, p<0.05). There was no difference in apoptosis, assessed asbefore by TUNEL and activated caspase-3, between nanoparticle injectedtumors in the presence or absence of the RF magnetic field (FIGS. 10Eand F)(TUNEL: control 56.4±9.8 cells, RF 48.8±8.9 cells. Active Caspase3: control 199.8±27.4 cells, RF 168±24.4 cells)

To ensure the effects on blood glucose were not the result ofnon-specific release of insulin due to thermal effects of nanoparticleson the tumor, the in vivo study was repeated using PC12 cellsestablished to stably express calcium regulated furin sensitive insulinalone without TRPV1His and it was found that there was no significanteffect on blood glucose in mice injected with PBS or nanoparticles withRF magnetic field application (FIG. 12). Nor was there a cumulativechange in blood glucose (FIG. 10B) (NF Ins AUC (0-120 min): PBS treated1851±1126 mgdl-1 min, nanoparticle treated 1281±1758 mgdl-1 min) or inplasma insulin levels (FIG. 10C) (Plasma insulin: PBS (−30 min)1.77±0.310 μIU/ml, PBS (30 min) 1.31±0.150 μIU/ml, nanoparticles (−30min) 1.2±0.170 μIU/ml, nanoparticles (30 min) 1.17±0.230 μIU/ml).Finally, there was no difference in proinsulin mRNA expression betweennanoparticle-injected NF Ins tumors with or without RF magnetic fieldtreatment (FIG. 10D) (Control: 1.0±0.4 relative insulin gene expression,RF treated: 1.0±0.2 relative insulin gene expression). Intratumoraltemperatures did not differ between RF treated PC12 TRPV1His-Ins andPC12 Ins tumors (FIGS. 12B and 12C). Thus the release of insulin andlowering of blood glucose requires the presence of the particles, themodified TRPV1 channel and the NFAT-insulin construct. These findingsdemonstrate that magnetic field heating of nanoparticles coupled toTRPV1 activation remotely and specifically regulates proinsulin mRNAsynthesis and insulin release to reduce blood glucose.

The development of a cell based system for remotely activating cells andits utility for lowering blood glucose in mice is demonstrated herein.There have been relatively few advances in the therapies available fordiseases caused by tissue loss—the mainstay of treatment remainspharmacological replacement often with marked side effects. Remotemodulation of cell activity and downstream functions such as geneexpression, protein synthesis and secretion combined with developmentsin stem cell therapy offers the prospect of more physiological therapiesin the form of engineered autografts. Nanoparticle dependent calciumentry could be used to regulate not only hormone release but alsofunctions such as neural activity or muscle contraction.

The system described herein can be further modified to achievecombinatorial activation of different cells. Cell populations expressingTRPV1 engineered to incorporate unique epitope tags could be decoratedwith different nanoparticles designed to heat in response to distinct RFfrequencies allowing combinatorial cell activation and peptide release.For example, gold nanoparticles, could be labeled with a second antibodyto a different epitopes on the modified TRPV1 channel in cellsengineered to express glucagon. This would enable one to either lower orraise blood glucose depending on the ambient blood glucoseconcentration.

These studies provide a platform for developments using different typesof nanoparticles with enhanced characteristics. For example, refinementsin nanoparticle structures could be used to depolarize cells without theneed for a TRP channel; this advance would allow the direct activationof cells using nanoparticles with antibodies directed against specificcell surface epitopes. It may also be possible to engineer cells thatexpress nanoparticles intracellularly thus obviating the need forinjecting particles.

The present invention is not to be limited in scope by the specificembodiments described herein which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the claims. Variouspublications are cited herein, the contents of which are herebyincorporated, by reference, in their entireties.

What is claimed:
 1. A method to remotely modulate the activity of a cell type of interest, the method comprising: (a) administering to a cell population nanoparticles selective for a temperature sensitive channel within the cell type of interest; and (b) applying a radiofrequency field to remotely activate the nanoparticles, wherein said activation of the nanoparticles results in modulation of the activity of the cell type of interest.
 2. The method of claim 1, wherein the activation of the nanoparticles results in an increase in the temperature of the particles and the temperature sensitive channel.
 3. The method of claim 2, wherein the temperature sensitive channel is selected from the group consisting of TRPV1, TRPV2, TRPV3, TRPM8, chimeric TRP channels, and tandem pore domain potassium channels.
 4. The method of claim 1, wherein the cell type of interest is a stem cell.
 5. The method of claim 1, wherein the nanoparticles comprise metallic or metal oxide nanoparticles.
 6. A method to remotely modulate the activity of a cell type of interest in a subject, the method comprising: (a) administering to the subject nanoparticles selective for a temperature sensitive channel within the cell type of interest; and (b) applying a radiofrequency field to remotely activate the nanoparticles, wherein said activation of the nanoparticles results in modulation of the activity of the cell type of interest in a subject.
 7. The method of claim 6, wherein activation of the nanoparticles results in an increase in the temperature of the particles and the temperature sensitive channel.
 8. The method of claim 7, wherein the temperature sensitive channel is the TRPV1 channel.
 9. The method of claim 6, wherein the cell type of interest is a stem cell.
 10. The method of claim 6, wherein the nanoparticles comprise metallic or metal oxide nanoparticles.
 11. A method to modulate the activity of a cell type of interest in a subject, the method comprising steps: (a) administering to the subject modified cells of interest that comprise nanoparticles that are selective for a temperature sensitive channel within the cell type of interest; and (b) applying a radiofrequency field to remotely activate the nanoparticles, wherein said activation of the nanoparticles results in modulation of the activity of the cell type of interest in a subject.
 12. The method of claim 11, wherein activation of the nanoparticles results in an increase in the temperature of the particles and the temperature sensitive channel.
 13. The method of claim 12, wherein the temperature sensitive channel is the TRPV1 channel.
 14. The method of claim 11, wherein the cell type of interest is a stem cell.
 15. The method of claim 11, wherein the nanoparticles comprise metallic or metal oxide nanoparticles.
 16. The method of claim 11, wherein the nanoparticle is an intracellularly expressed nanoparticle.
 17. The method of claim 16, wherein the nanoparticle is ferritin.
 18. The method of claim 16, wherein the nanoparticle is a ferritin fusion protein.
 19. The method of claim 1, wherein the temperature sensitive channel comprises TRPV1.
 20. The method of claim 3, wherein the tandem pore domain potassium channel comprises: TREK1, TREK2, or TASK.
 21. The method of claim 1, wherein the temperature sensitive channel comprises a fusion protein.
 22. The method of claim 21, wherein the fusion protein comprises a tag.
 23. The method of claim 22, wherein the tag comprises: a nanobody peptide, His tag, or biotin acceptor protein.
 24. The method of claim 1, wherein the nanoparticles comprise: ferritin, MagA, ceruloplasmin, or transferrin.
 25. The method of claim 1, wherein the nanoparticles comprise a targeting moiety.
 26. The method of claim 25, wherein the targeting moiety comprises: antibody, streptavidin, or peptide. 